Method of Treating and Preventing Infectious Diseases via Creation of a Modified Viral Particle with Immunogenic Properties

ABSTRACT

The present invention relates to a method for reducing the occurrence and severity of infectious diseases, especially infectious diseases in which lipid-containing infectious organisms are found in biological fluids, such as blood. The present invention employs solvents useful for extracting lipids from the lipid-containing infectious organism, thereby reducing the infectivity of the infectious organism. The present invention uses optimal solvent systems such that the lipid envelope around the viral particle is dissolved while the viral particle remains intact, resulting in a modified viral particle. The present invention also provides an autologous vaccine composition, comprising a lipid-containing infectious organism, treated with solvents to reduce the lipid content of the infectious organism, combined with a pharmaceutically acceptable carrier. The vaccine composition is administered to an animal or a human to provide protection against the lipid-containing infectious organism. The present invention further provides a simple, inexpensive and easy to use kit for delipidating fluids and for delipidation of lipid-containing organisms in a fluid.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. non-provisionalpatent application Ser. No. 10/311,679 filed Dec. 18, 2002, which is aUS national phase from PCT patent application number PCT/IB01/01099filed Jun. 21, 2001, which claims the benefit of Australian patentapplication PQ8469 filed Jun. 29, 2000 and PCT patent application numberPCT/AU00/01603 filed Dec. 28, 2000. The present application also claimsthe benefit of U.S. provisional patent application Ser. No. 60/390,066filed Jun. 20, 2002.

FIELD OF THE INVENTION

The present invention relates to a delipidation method employing asolvent system useful for extracting lipids from a virus, therebycreating a modified viral particle. The solvent system of the presentinvention is optimally designed such that upon delipidation of thevirus, the viral particle remains substantially intact. By dissolvingthe lipid envelope surrounding the viral particle using the method ofthe present invention, the resultant modified viral particle has exposedantigens (or epitopes), which foster and promote antibody production.The resulting modified viral particle of the present invention initiatesa positive immunogenic response in the species into which it isre-introduced. The present invention can be applied to delipidatingviruses from a specific patient for future reintroduction into thepatient, to delipidating stock viruses, or non-patient specific viruses,for use as a vaccine, or to delipidating and combining both non-patientspecific viruses and patient specific viruses to create a therapeuticcocktail.

BACKGROUND OF THE INVENTION Introduction

Viruses, of varied etiology, affect billions of animals and humans eachyear and inflict an enormous economic burden on society. Many virusescontain lipid as a major component of the membrane that surrounds them.Viruses affect animals and humans causing extreme suffering, morbidity,and mortality. These viruses travel throughout the body in biologicalfluids such as blood, peritoneal fluid, lymphatic fluid, pleural fluid,pericardial fluid, cerebrospinal fluid, and in various fluids of thereproductive system. Fluid contact at any site promotes transmission ofdisease. Other viruses reside primarily in different organ systems andin specific tissues, proliferate and then enter the circulatory systemto gain access to other tissues and organs at remote sites. If the bodydoes not exhibit a positive immune response against these pathogens,they infect many cell types within the body, inhibiting these cells fromperforming their normal functions.

The human immune system is composed of various cell types thatcollectively protect the body from different viruses. The immune systemprovides multiple means for targeting and eliminating foreign elements,including humoral and cellular immune responses, participating primarilyin antigen recognition and elimination. An immune response to foreignelements requires the presence of B-lymphocytes (B cells) orT-lymphocytes (T cells) in combination with antigen-presenting cells(APC), which are usually macrophage or dendrite cells. The APCs arespecialized immune cells that capture antigens. Once inside an APC,antigens are broken down into smaller fragments called epitopes—theunique markers carried by the antigen surface. These epitopes aresubsequently displayed on the surface of the APCs and are responsiblefor triggering an antibody response in defense of the infection.

In a humoral immune response, when an APC displaying antigens (in theform of unique epitope markers) foreign to the body are recognized, Bcells are activated, proliferating and producing antibodies. Theseantibodies specifically bind to the antigens present on the APC andblock their ability to further infect cells. After the antibodyattaches, the APC engulfs the entire antigen and kills it. This type ofantibody immune response is primarily involved in the prevention ofviral infection.

In a cellular immune response, on recognizing the APC displaying aforeign antigen, the T cells are activated. There are two steps in thecellular immune response. The first step involves activation ofcytotoxic T cells (CTL) or CD8+ T killer cells that proliferate and killtarget cells that specifically present antigens presented by APC. Thesecond involves helper T cells (HTL) or CD4+ T cells that regulate theproduction of antibodies and the activity of CD8+ cells. The CD4+ Tcells provide growth factors to CD8+ T cells that allow them toproliferate and function efficiently.

Certain infective pathogens are deemed “chronic” due to their structure.For example, some viruses are able to evade an immune response becauseof their ability to hide some of their antigens from the immune system.Viruses contain an outer envelope made up of lipids and fats derivedfrom the host cell membrane during the budding process. Viruses arecomprised of virions, non-cellular infectious agents consisting of asingle type of nucleic acid (either RNA or DNA), surrounded by a proteincoat. The outer protein covering of viruses is called a capsid, made upof repeating subunits called capsomeres.

Since viruses are non-metabolic, they only reproduce within living hostcells. The virus codes the proteins of the viral envelope while the hostcell codes the lipids and carbohydrates. Therefore, the lipid andcarbohydrate content within a given viral envelope is dependent on theparticular host. The enveloped viral particles therefore partially adoptthe identity of the host cell, via lipid and carbohydrate content, andare able to conceal antigens associated with them, which would normallyhave initiated an immune response. Instead, the viral particle confusesthe host immune system by presenting it with an antigenic complex thatcontains components of host tissues, and is perceived by the host immunesystem as partly “self” and partly “foreign”. An immune response thatdestroys the antigenic complex containing host tissue elements can endup destroying host cells leading to severe autoimmune disease. Theimmune system is forced to produce the “compromise”, ineffectiveantibodies which do not destroy the viral particles, allowing them toproliferate and slowly cause severe damage to the body, while destroyinghost cells.

Recent epidemics affecting the immune system include acquired immunedeficiency syndrome (AIDS), believed to be caused by the humanimmunodeficiency virus (HIV). Related viruses affect animal species, forexample, simians and felines (SIV and FIV, respectively). Other majorviral infections include, but are not limited to, meningitis,cytomegalovirus, and hepatitis in its various forms.

Current Methods of Treatment

One prior art method of treating viruses of varied etiology is via drugtherapy. Most anti-viral drug therapies are directed to preventing orinhibiting viral replication and appear to focus on the initialattachment of the virus to the T4 lymphocyte or macrophage, thetranscription of viral RNA to viral DNA and the assembly of new virusduring reproduction. The high mutation rate of the virus, especially inthe case of HIV, is a major difficulty with existing treatments becausethe various strains become resistant to anti-viral drug therapy.Furthermore, anti-viral drug therapy treatment may cause the evolutionof resistant strains of the virus. Other drawbacks to drug therapies arethe undesirable side effects and patient compliance requirements. Inaddition, many individuals are afflicted with multiple viral infectionssuch as a combination of HIV and hepatitis. Such individuals requireeven more aggressive and expensive drug regimens to counteract diseaseprogression, which in turn cause greater side effects and a greaterlikelihood of multiple drug resistance.

Also known in the prior art is prevention of disease via the use ofvaccinations. Vaccines have been singularly responsible for conferringimmune response against several human pathogens. They are designed tostimulate the immune system to protect against various viral infections.In general, a vaccine is produced from an antigen, isolated or producedfrom the disease-causing microorganism, which can elicit an immuneresponse. When a vaccine is injected into the blood stream as apreventive measure to create an effective immune response, the B cellsin the blood stream perceive the antigens contained by the vaccine asforeign or ‘non-self’ and respond by producing antibodies, which bind tothe antigens and inactivate them. Memory cells are thereby produced andremain ready to mount a quick protective immune response againstsubsequent infection with the same disease-causing agent. Thus when aninfective pathogen containing similar antigens as the vaccine enters thebody, the immune system will recognize the protein and instigate aneffective defense against infection.

The current methods of vaccination do have drawbacks, making them lessthan optimally desirable for immunizing individuals against particularpathogens, especially HIV. The existing vaccine strategies aim to exposethe body to the antigens associated with infective pathogens so that thebody builds an immune response against these pathogens. For example,hepatitis B and HIV pathogens are able to survive and proliferate in thehuman body despite having an effective immune response. One explanationoffered in the prior art is that the antigens of these microorganismschange constantly so the antibodies produced in response to a particularantigen are no longer effective when the antigen mutates. The AIDS virusis believed to undergo this antigenic variation. Although antigenicvariation has been addressed via the attempted use of combination drugsor antigens, no prior art vaccine has succeeded in addressing chronicinfections such as HIV.

Another approach to treating viruses of varied etiology is to inactivatethe virus. Prior art methods of inactivating viruses using chemicalagents have relied on organic solvents such as chloroform orglutaraldehyde. Although viral inactivation is effective in reducingviral load of a patient and treating contaminated blood to be used inblood transfusions, it does have problems. For example, inactivation ofa virus does not provide a protective immune response against viralinfection. In addition, it is largely geared towards denaturing viralproteins, thereby destroying the structure of the viral particle.

Drug therapy, as described above only provides a temporary solution toviral infectivity and works only to decrease the viral load of apatient. Chemical inactivation of the virus works to temporarilydecrease viral infectivity; however, once cells replicate the level ofinfectivity will increase again. Moreover, these destruction-typeprocesses lead to total cell death and do not initiate or promote apositive immunogenic response in the patient. In sum, prior art methodshave largely focused on destroying, yet not suitably modifying, viralparticles.

Current Methods of Manufacture of Viral Treatments and Medicaments ViralInactivation (or Chemical Kill)

Described in the prior art are methods of treating viral particles withorganic solvents and high temperatures thus dissolving the lipidenvelopes and subsequently inactivating the virus. In those methods,blood is withdrawn from the patient and separated into two phases—thefirst phase including red cells and platelets and the second phasecontaining plasma, white cells, and cell-free virus (virion). The secondphase is treated with an organic solvent, thereby killing the infectedcells and virions, and subsequently reintroduced into the patient. Inaddition to dissolving the lipid envelope of the virus, the high organicsolvent concentrations cause cell death and damage to the antigens.Essentially, this method results in a “chemical kill” of the cell.

Glutaraldehyde is one such solvent whereby cell inactivation is achievedas known by those of ordinary skill in the art by fixation with a dilutesolution of glutaraldehyde at about 1:250. Although treating the viruswith glutaraldehyde effectively delipidates the virus, it also destroysthe core. Destruction of the core is not desirable for producing amodified viral particle useful for inducing an immune response in arecipient.

Chloroform is another such solvent. Chloroform, however, denatures manyplasma proteins and is not suitable for use with biological fluids,which will be reintroduced into the animal or human. These plasmaproteins deleteriously affected by chloroform serve important biologicalfunctions including coagulation, hormonal response, and immune response.These functions are essential to life and thus damage to these proteinsmay have an adverse effect on a patient's health, possibly leading todeath.

Other solvents or detergents such as B-propiolactone, TWEEN-80, anddialkyl or trialkyl phosphates have been used, either alone or incombination. Many of these methods, especially those involvingdetergents, require tedious procedures to ensure removal of thedetergent before reintroduction of the treated plasma sample into theanimal or human. Further, many of the methods described in the prior artinvolve extensive exposure to elevated temperature in order to kill freevirus and infected cells. Elevated temperatures have deleterious effectson the proteins contained in biological fluids, such as plasma.

Current Methods of Manufacturing Vaccines

To date, several manufacturing methods have been employed in search forsafe and effective vaccines for immunizing individuals against infectivepathogenic agents. To protect an individual from a specific pathogenicinfection, a target protein or antigen associated with the infectivepathogen is administered to the individual. This includes presenting theprotein as part of a non-infective (inactivated) or less infective(attenuated) agent or as a discreet protein composition. Known to one ofordinary skill in the art are the following different types of vaccines:live attenuated vaccines, whole inactivated vaccines, DNA vaccines,combination vaccines, recombinant vaccines, live recombinant vectorvaccines, virus like particles and synthetic peptide vaccines.

In live attenuated vaccines, the viruses are rendered less pathogenic tothe host, either by specific genetic manipulation of the virus genome orby passage in some type of tissue culture system. In order to achievegenetic manipulation, an inessential gene is deleted or one or moreessential genes in the virus are partially damaged. Upon geneticmanipulation, the viral particles become less virulent yet retainantigenic features. Live attenuated vaccines can also be used as“vaccine vectors” for other genes, wherein they act as carriers of genesfrom a second virus (or other pathogen) against which protection isrequired. Attenuated vaccines (less infective and not inactivated),however, pose several problems. First, it is difficult to ascertain whenthe attenuated vaccine is no longer pathogenic. The risk of viralinfection from the vaccine is too great to properly test for effectiveattenuation. In addition, attenuated vaccines carry the risk ofreverting into a virulent form of the pathogen.

Whole inactivated vaccines are known in the art for immunizing againstinfection by introducing killed or inactivated viruses to introducepathogen proteins to an individual's immune system. The administrationof killed or inactivated pathogen, via heat or chemical means, into anindividual introduces the pathogen to the individual's immune system ina non-infective form thereby instigating an immune response defense.Wholly inactivated vaccines provide protection by directly generatingcellular and humoral immune responses against the pathogenic immunogens.There is little threat of infection, because the viral pathogen iskilled or otherwise inactivated.

Subunit vaccines are yet another form of vaccination well known to oneof ordinary skill in the art. These consist of one or more isolatedproteins derived from the pathogen. These proteins act as targetantigens against which an immune response is exhibited. The proteinsselected for the subunit vaccine are displayed by the pathogen so thatupon infection of an individual by the pathogen, the individual's immunesystem recognizes the pathogen and instigates an immune response.Subunit vaccines are not whole infective agents and are thereforeincapable of becoming infective. Subunit vaccines are the basic ofAIDSVAX, the first vaccine for HIV being tested for effectiveness inhumans and which contains a portion of HIV's outer surface (envelope)protein, called gp120.

DNA vaccine is another type known in the art and uses actual geneticmaterial of pathogens. In addition, synthetic peptide vaccines are madeup of parts of synthetic, chemically engineered HIV proteins calledpeptides. They comprise portions of HIV proteins chosen specifically toachieve an anti-HIV immune response. Also mentioned in the prior art arecombination vaccines that, when used in conjunction with one another,generate a broad spectrum of immune responses. One example of acombination vaccine is SHIV, which is a synthetic vaccine made from theHIV envelope and SIV core.

What is needed is a therapeutic method and system for providing patientswith patient-specific antigens capable of initiating a protective immuneresponse. Accordingly, what is needed is a simple, effective method thatdoes not appreciably denature or extract plasma proteins from thebiological sample being treated. What is also needed is an effectivedelipidation process via which a viral particle is modified, rather thandestroyed, thereby both reducing and/or eliminating infectivity of theviral particle and invoking a patient specific, autologous immuneresponse to reduce viral infection and prevent further infection.

What is also needed is an effective means to immunize individualsagainst viral pathogen infection that is unique to the individual due toviral mutations. Preferably the means would elicit a broad, biologicallyactive protective immune response with minimized risk of infecting theindividual.

SUMMARY OF THE INVENTION

The present invention solves the problems described above by providing asimple, effective and efficient method for treating and preventing viralinfection. The method of the present invention affects the lipidenvelope of a virus by utilizing an efficient solvent system, which doesnot denature or destroy the virus. The present invention employs anoptimal solvent and energy system to create, via delipidation, anon-synthetic, host-derived modified viral particle that has its lipidenvelope at least partially removed, generating a positive immunologicresponse in a patient, thereby providing that patient with some degreeof protection against the virus.

The present invention is also effective in producing an autologous,patient-specific therapeutic vaccine against the virus, by treating abiological fluid containing the virus such that the virus is present ina modified form, but no longer infectious and such that an immuneresponse is initiated upon reintroduction of the fluid into the patient.To create the vaccine, a biological fluid (for example, blood) isremoved from the patient, the plasma is separated from the blood andtreated to isolate the virus, and the virus is delipidated using anoptimal solvent system. A lipid-containing virus, treated in this mannerin order to reduce its infectivity and create a modified viral particle,is administered to a recipient, such as an animal or a human, togetherwith a pharmaceutically acceptable carrier in order to initiate animmune response in the animal or human and create antibodies that bindthe exposed epitopes of the modified viral particle. Adjuvants may alsobe administered with the modified viral particle in the pharmaceuticallyacceptable carrier.

Thus an effective method is presented, by which new vaccines can bedeveloped out of lipid containing viruses by removing lipid from thelipid envelope and exposing antigens hidden within the lipid envelope orbeneath the surface of the lipid envelope, in turn generating a positiveimmune response when re-introduced into the patient.

The present invention provides a modified viral particle comprising atleast a partially delipidated viral particle, wherein the partiallydelipidated viral particle initiates a positive immune response in ananimal or human patient and incites protection against an infectiousorganism in the animal or the human patient.

The present invention provides a method for creating a modified viralparticle comprising the steps of: receiving a plurality of viralparticles, each having a viral envelope, in a fluid; exposing the viralparticles to a delipidation process; and, partially delipidating theviral particles wherein the delipidation process at least partiallyremoves the viral envelopes to create the modified viral particle andwherein the modified viral particle is capable of provoking a positiveimmune response in a patient.

The present invention also provides an antigen delivery vehicle and amethod for creating an antigen delivery vehicle comprising the steps of:receiving a plurality of viral particles, each having a viral envelope,in a fluid; exposing the viral particles to a delipidation process; and,partially delipidating the viral particles to create modified viralparticles that act as antigen delivery vehicles, wherein thedelipidation process at least partially removes the viral envelopes toexpose at least one antigen and wherein the at least one antigen iscapable of provoking a positive immune response in a patient.

The modified viral particles of the present invention comprise at leasta partially delipidated viral particle, wherein the partiallydelipidated viral particle is produced by exposing a non-delipidatedviral particle to a delipidation process and wherein the partiallydelipidated viral particle comprises at least one exposed patientspecific antigen that was not exposed in the non-delipidated viralparticle.

The present invention also provides a vaccine composition, comprising atleast a partially delipidated viral particle having patient-specificantigens and a pharmaceutically acceptable carrier, wherein thepartially delipidated viral particle is capable of provoking a positiveimmune response when the composition is administered to a patient.

The present invention also provides a method for making a vaccinecomprising: contacting a lipid-containing viral particle in a fluid witha first organic solvent capable of extracting lipid from thelipid-containing viral particle; mixing the fluid and the first organicsolvent for a time sufficient to extract lipid from the lipid-containingviral particle; permitting organic and aqueous phases to separate; andcollecting the aqueous phase containing a modified viral particle withreduced lipid content wherein the modified viral particle is capable ofprovoking a positive immune response when administered to a patient.

The present invention also provides a method to protect an animal or ahuman against an infectious viral particle comprising administering tothe animal or the human an effective amount of a composition comprisinga modified viral particle, wherein the modification comprises at leastpartial removal of a lipid envelope of the infectious viral particle,and a pharmaceutically acceptable carrier, wherein the amount iseffective to provide a protective effect against infection by theinfectious viral particle in the animal or the human.

The present invention also provides a method for provoking a positiveimmune response in an animal or human having a plurality oflipid-containing viral particles, comprising the steps of: obtaining afluid containing the lipid-containing viral particles from the animal orthe human; contacting the fluid containing the lipid-containing viralparticles with a first organic solvent capable of extracting lipid fromthe lipid-containing viral particles; mixing the fluid and the firstorganic solvent: permitting organic and aqueous phases to separate;collecting the aqueous phase containing modified viral particles withreduced lipid content; and introducing the aqueous phase containing themodified viral particles with reduced lipid content into the animal orthe human wherein the modified viral particles with reduced lipidcontent provoke a positive immune response in the animal or the human.

The present invention also provides a method for treating a viralinfection in an animal or human patient comprising: removing bloodcontaining a plurality of lipid-containing infectious viral particlesfrom the animal or the human; obtaining plasma from the blood, theplasma containing the lipid-containing infectious viral particles;contacting the plasma containing the lipid-containing infectious viralparticles with a first organic solvent capable of extracting lipid fromthe lipid-containing infectious viral particles to produce modifiedviral particles having reduced lipid content; mixing the plasma and thefirst organic solvent; permitting organic and aqueous phases toseparate; collecting the aqueous phase containing the modified viralparticles; and introducing the aqueous phase containing the modifiedviral particles into the animal or the human wherein the modified viralparticles have at least one exposed patient-specific antigen that wasnot exposed in the plurality of lipid-containing infectious viralparticles.

As shown below, the characteristics of the modified viral particle areexhibited in experimental data, showing mice having a positiveimmunogenic response when vaccinated as compared with a whollyinactivated vaccine. In addition, data exhibiting protein recoveryindicate retention of the structural integrity of the viral particle,removing only its lipid-containing envelope.

Fluids which may be treated with the method of the present inventioninclude but are not limited to the following: plasma; serum; lymphaticfluid; cerebrospinal fluid; peritoneal fluid; pleural fluid; pericardialfluid; various fluids of the reproductive system including but notlimited to semen, ejaculatory fluids, follicular fluid and amnioticfluid; cell culture reagents such as normal sera, fetal calf serum orserum derived from any other animal or human; and immunological reagentssuch as various preparations of antibodies and cytokines.

The method of the present invention may be used to treat virusescontaining lipid in the viral envelope. Preferred viruses to be treatedwith the method of the present invention include the variousimmunodeficiency viruses including but not limited to human (HIV) andsubtypes such as HIV-1 and HIV-2, simian (SIV), feline (FIV), as well asany other form of immunodeficiency virus. Other preferred viruses to betreated with the method of the present invention include but are notlimited to hepatitis in its various forms. Another preferred virustreated with the method of the present invention is the bovinepestivirus. It is to be understood that the present invention is notlimited to the viruses provided in the list above. Additional specificviruses are described in the detailed description of this application.All viruses containing lipid, especially in their viral envelope, areincluded within the scope of the present invention.

The present invention also provides a simple, inexpensive and easy touse kit for delipidating fluids and lipid-containing viruses withinfluids and to create modified viral particles. This kit may be used invarious situations, such as in the field, in a clinic, by a physician inan emergency situation, in a laboratory or elsewhere. Preferred fluidsinclude biological fluids and culture medium. A preferred biologicalfluid is plasma.

The kits of the present invention may be used to process plasma from apatient. In a preferred embodiment, the plasma contains lipid-containingvirus. This delipidated plasma containing modified viral particles maybe stored in a blood bank for subsequent autologous or heterologousadministration. The kits of the present invention may be used to processplasma from a patient and then administer the delipidated plasma,containing modified viral particles, to the patient. The kits of thepresent invention may be used to process other fluids containing lipidsor lipid-containing viruses, such as culture media and cells cultured inmedia. The modified viral particles produced with these kits may becombined with a pharmaceutically acceptable carrier, and optionally anadjuvant, and used as vaccines by administration to an animal or humanto cause an immune response to epitopes on or in the modified viralparticles.

The kits of the present invention generally comprise containers used fordifferent purposes. A first container generally contains one or morefirst extraction solvents. This first container contains means forremoving the first extraction solvent from the container. Such means maybe a component of the first container or a separate component adapted tofunction with the first container. Such means include, but are notlimited to, any type of cap, spout, funnel, penetrable seal, penetrablediaphragm, tube, pipette, or other means known to one skilled in the artfor removing the one or more first extraction solvents or forintroducing a fluid containing lipid or lipid-containing virus into thefirst container. A second container contains the fluid containing lipidsor lipid-containing virus to be delipidated.

In one embodiment, a third container is used for contacting or mixingthe fluid containing lipids or lipid-containing virus to be delipidatedand the first extraction solvent. Mixing can occur through agitation,inversion, shaking, or other means to agitate the third container to adegree sufficient to mix the fluid and the first extraction solvent.After the mixing step, the first extraction solvent containing thedissolved lipids from the fluid or from the lipid-containing organismsseparates from the fluid. At this point, the delipidated fluid may beremoved through any means such as pouring, decanting, pipetting,applying a vacuum connected to a tube or pipette, or any other meansknown to one of ordinary skill in the art of removing separated fluids.

A fourth container optionally receives the delipidated fluid andmodified viral particles from the third container. Alternatively, thedelipidated fluid and modified viral particles are administered to thepatient through a tube, catheter, an intravenous line, an intraarterialline or other means without introduction into a fourth container.

In one embodiment the first container contains sufficient volume withinit to receive the fluid containing lipids or lipid-containing virus tobe delipidated. In this embodiment, mixing of the first extractionsolvent and the fluid containing lipids or lipid-containing virus to bedelipidated occurs within the first container. In this embodiment, aseparate container for mixing the fluid and the first extractionsolvent, referred to as the third container above, is not required.After mixing occurs, the first extraction solvent containing thedissolved lipids separates from the delipidated fluid. At this point,the delipidated fluid may be introduced into another container,analogous to the fourth container described above for subsequentintroduction into a patient or for optional additional extraction of thefirst extraction solvent with a second extraction solvent.

In another embodiment, when a second extraction solvent is optionallyemployed to assist in removal of the first extraction solvent, a fifthcontainer is included which contains the second extraction solvent. Thissecond extraction solvent may be added to the mixture described above inthe third container, mixed and then permitted to separate from thedelipidated fluid. Alternatively, the second extraction solvent may beadded to the fourth container described above, mixed and then permittedto separate from the delipidated fluid if additional removal of residualfirst extraction solvent is desired. In yet another alternativeembodiment, the second extraction solvent may be added to the firstcontainer described above containing the mixture of the fluid and thefirst extraction solvent, mixed and then permitted to separate from thedelipidated fluid, if mixing of the fluid and the first extractionsolvent, separation and additional extraction of the first extractionsolvent using a second extraction solvent are all performed in the firstcontainer. The containers described above may be graduated for easydetermination of volume within a container.

Accordingly, it is an object of the present invention to provide amethod for treating lipid containing virus in order to create modifiedviral particles.

It is another object of the present invention to provide a method fortreating or preventing viral disease by administering modified viralparticles to a patient.

Another object of the present invention is to provide a method fortreating a biological fluid in order to reduce or eliminate theinfectivity of infectious viral organisms contained therein.

It is further an object of the present invention to provide a method fortreatment of lipid-containing viruses within a fluid, which minimizesdeleterious effects on proteins contained within the fluid, therebycreating a modified viral particle with properties that are capable ofinitiating a positive immune response in a patient.

It is a further object of the present invention to provide a method fortreatment of lipid-containing viruses within a fluid, which minimizesdeleterious effects on proteins contained within the fluid, therebycreating a modified viral particle with patient-specific antigens.

It is another object of the present invention to provide a method forreducing the infectivity of viruses, wherein the method does not employelevated temperatures, chloroform, detergents, or trialkyl phosphates.

It is another object of the present invention to provide a method forreducing the infectivity of viruses, wherein the method exposesantigenic determinants on the modified viral particle.

It is a farther object of the present invention to completely orpartially delipidate the viral particle, wherein the viral particlescomprise immunodeficiency virus, hepatitis in its various forms, or anyother lipid-containing virus, thereby creating a modified viralparticle.

It is a further object of the present invention to completely orpartially delipidate the viral particle, wherein the viral particlescomprise immunodeficiency virus, hepatitis in its various forms, or anyother lipid-containing virus, while retaining the structural proteincore of the virus.

It is another object of the present invention to provide a method forreducing the infectivity of viruses, wherein the newly formed viralparticle can be used as an antigen delivery vehicle.

Yet another object of the present invention is to treat infectiousorganisms with the method of the present invention in order to reducetheir infectivity and provide a vaccine comprising a delipidated,modified viral particle which may be administered to an animal or ahuman together with a pharmaceutically acceptable carrier and optionallyan immunostimulant compound, to prevent or minimize clinicalmanifestation of disease in a patient following exposure to the virus.

Yet another object of the present invention is to treat infectiousorganisms with the method of the present invention in order to reducetheir infectivity and provide a vaccine comprising a delipidated,modified viral particle which may be administered to an animal or ahuman together with a pharmaceutically acceptable carrier and optionallyan immunostimulant compound, to initiate a positive immunogenic responsein the animal or human.

It is another specific object of the present invention to provide ananti-viral vaccine.

It is a further specific object of the present invention to lessen theseverity of a disease caused by a lipid-containing virus in an animal orhuman receiving a vaccine comprising a composition comprising a virustreated with the method of the present invention in a pharmaceuticallyacceptable carrier.

It is another object of the present invention to combine delipidatedviral particles having patient specific antigens with delipidated stockviral particles to create a therapeutic cocktail for the treatment ofdiseases.

Accordingly it is an object of the present invention to provide aninexpensive and easy to use kit for removal of lipids from fluids andinfectious organisms, preferably biological fluids, plasma, or culturemedium.

Another object of the present invention is to provide an inexpensive andeasy to use kit for removal of lipids from lipid-containing viruses tocreate modified viral particles.

These and other features and advantages of the present invention willbecome apparent after review of the following drawings and detaileddescription of the disclosed embodiments. Various modifications to thestated embodiments will be readily apparent to those of ordinary skillin the art, and the disclosure set forth herein may be applicable toother embodiments and applications without departing from the spirit andscope of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and form a part ofthe specification, illustrate preferred embodiments of the presentinvention.

FIG. 1 is a schematic diagram of an embodiment of a kit of the presentinvention containing a first container 10 with a screw cap 15,containing first extraction solvent 20, and plasma 30, and a secondcontainer 50 with a tube 60 leading from an opening 70, the tube 60being connected to a needle 62.

FIG. 2 is a schematic diagram of an embodiment of a kit of the presentinvention containing a first container 10 with a screw cap 15,containing first extraction solvent 20, a second container 50 with ascrew cap 55 and containing plasma 30, a third container 70, with ascrew cap 75 for mixing the first extraction solvent 20 and plasma 30 toform mixture 72, and a fourth container 80 with a screw cap 85 forstoring delipidated plasma 82.

FIG. 3 incorporates the elements of FIG. 2 and further provides a fifthcontainer 90 with a screw cap 95 containing a second extraction solvent92 and a sixth container 100 for storing delipidated plasma 102 withreduced levels of residual first extraction solvent, with a tube 110leading from an opening 105, the tube 110 being connected to a needle112.

FIG. 4 is a schematic representation of an HIV viral particle showingthe lipid containing envelope (LE) or bilayer derived from a host cell,the capsid (C), nuclear material (NM) such as RNA, surface glycoproteins(GP) such as gp120 and gp41, transmembrane proteins (TMP), p17 matrixprotein, and capsid proteins (CP) such as p24.

DETAILED DESCRIPTION OF THE INVENTION Definitions

By the term “fluid” is meant any fluid containing an infectiousorganism, including but not limited to, a biological fluid obtained froman organism such as an animal or human. Preferred infectious organismstreated with the method of the present invention are viruses. Suchbiological fluids obtained from an organism include but are not limitedto plasma, serum, cerebrospinal fluid, lymphatic fluid, peritonealfluid, follicular fluid, amniotic fluid, pleural fluid, pericardialfluid, reproductive fluids and any other fluid contained within theorganism. Other fluids may include laboratory samples containinginfectious organisms suspended in any chosen fluid. Other fluids includecell culture reagents, many of which include biological compounds suchas fluids obtained from living organisms, including but not limited to“normal serum” obtained from various animals and used as growth mediumin cell and tissue culture applications.

By the terms “first solvent” or “first organic solvent” “or firstextraction solvent” are meant a solvent, comprising one or moresolvents, used to facilitate extraction of lipid from a fluid or from alipid-containing biological organism. This solvent will enter the fluidand remain in the fluid until being removed. Suitable first extractionsolvents include solvents that extract or dissolve lipid, including butnot limited to alcohols, hydrocarbons, amines, ethers, and combinationsthereof. First extraction solvents may be combinations of alcohols andethers. First extraction solvents include, but are not limited ton-butanol, di-isopropyl ether (DIPE), diethyl ether, and combinationsthereof

The term “second extraction solvent” is defined as one or more solventsthat facilitate the removal of a portion of the first extractionsolvent. Suitable second extraction solvents include any solvent thatfacilitates removal of the first extraction solvent from the fluid.Second extraction solvents include any solvent that facilitates removalof the first extraction solvent including but not limited to ethers,alcohols, hydrocarbons, amines, and combinations thereof. Preferredsecond extraction solvents include diethyl ether and di-isopropyl ether,which facilitate the removal of alcohols, such as n-butanol, from thefluid. The term “de-emulsifying agent” is a second extraction solventthat assists in the removal of the first solvent which may be present inan emulsion in an aqueous layer.

The term “delipidation” refers to the process of removing at least aportion of a total concentration of lipids in a fluid or in alipid-containing organism. Lipid-containing organisms may be foundwithin fluids which may or may not contain additional lipids.

The terms “pharmaceutically acceptable carrier” or “pharmaceuticallyacceptable vehicle” are used herein to mean any liquid including but notlimited to water or saline, a gel, salve, solvent, diluent, fluidointment base, liposome, micelle, giant micelle, and the like, which issuitable for use in contact with living animal or human tissue withoutcausing adverse physiological responses, and which does not interactwith the other components of the composition in a deleterious manner.

The term “patient” refers to animals and humans in this application.

A Modified Viral Particle

Practice of the method of the present invention to reduce the lipidcontent of a virus creates a modified viral particle. These modifiedviral particles are immunogenic. The present methods expose epitopesthat are not usually presented to the immune system by untreated virus.A structural change occurs in the modified viral particles, and proteinson, in, or near the surface of the virus are modified such that aconformational change occurs. Some of these proteins may also separatefrom the modified viral particle. FIG. 4 is a schematic representationof HIV viral particle showing the lipid containing envelope or bilayerderived from a host cell, surface glycoproteins, transmembrane proteins,the capsid, capsid proteins and nuclear material. The delipidationprocess of the present invention modifies the viral particle. Themodified viral particle has a lower lipid content in the envelope,displays modified proteins, loses infectivity and is immunogenic.

Modified Viral Particle Resulting from Removal of Lipid fromLipid-Containing Organisms

As described above, methods of treating the viral particles with organicsolvents and use of high temperatures, thus dissolving the lipidenvelopes and subsequently inactivating the virus are well known in theprior art. In this method, blood is withdrawn from the patient andseparated into two phases—the first phase including red cells andplatelets and the second phase containing plasma, white cells, andcell-free virus (virion). The second phase is treated with an organicsolvent, thereby killing the infected cells and virions, andsubsequently reintroduced into the patient. In addition to dissolvingthe lipid envelope of the virus, however, the high organic solventconcentrations cause cell death and damage to the antigens. This methodresults in a “chemical kill” of the cell. Another drawback is thatelevated temperatures have deleterious effects on the proteins containedin biological fluids such as plasma. Glutaraldehyde is one such solventwhereby cell inactivation is achieved as known by those of ordinaryskill in the art by fixation with a dilute solution of glutaraldehyde atabout 1:250.

When a viral particle is sent through certain solvent systems, lipidswill be removed in the solvent because, when treated appropriately,lipids are soluble in certain solvent systems. Viruses are comprised ofvirions with the outer covering comprised of a protein coat, or capsid,as described above. Since viruses are non-metabolic, they only reproducewithin living host cells. The virus codes the proteins of the viralenvelope while the host cell codes the lipids and carbohydrates.Therefore, the lipid and carbohydrate within a given viral envelope isdependent on the particular host. The enveloped viral particlestherefore partially adopt the identity of the host cell and are able toconceal some antigens associated with the virus, which normally wouldhave initiated an immune response.

Instead, the viral particle confuses the host immune system bypresenting it with an antigenic complex that contains components of hosttissues, and is perceived by the host immune system as partly “self” andpartly “foreign”. An immune response that destroys the antigenic complexcontaining host tissue elements can destroy host cells leading to severeautoimmune disease. The immune system is forced to produce the“compromise”, ineffective antibodies which do not destroy the viralparticles, allowing them to proliferate and slowly cause severe damageto the body, while the host cells are destroyed.

Methods of the present invention can be used to solve this problembecause, by removing the lipid envelope of the virus, and keeping theviral particle intact, the method of the present invention exposesadditional antigens. The host immune system is forced to recognize theviral particle as wholly “foreign”. Using the method of the presentinvention, what is created is a modified viral particle in which theantigenic core remains intact, thereby using the epitopes of the actualviral particle to initiate a positive immunogenic response in thepatient into which it is reintroduced. In addition, the method of thepresent invention reduces the deleterious effect on the other plasmaproteins, measured by protein recovery, such that the plasma can bereintroduced into the patient.

In creating this modified viral particle what is also created is apatient-specific antigen that induces protection against the viralparticle in the species in which it is introduced. The method of thepresent invention creates an effective means to immunize individualsagainst viral pathogen infection and elicit a broad, biologically activeprotective immune response without risk of infecting the individual. Newvaccines may be developed from certain lipid containing viruses byremoving the lipid envelope and exposing antigens hidden beneath theenvelope, in turn generating a positive immune response. These“autologous vaccines” can be created by the at least partial removal ofthe lipid envelope using suitable solvent systems (one which would notdamage the antigens contained in the particle) exposing antigens and/orforcing a structural modification in the viral protein structures, whichwhen introduced into the body, would provoke an effective immuneresponse.

Infectious Organisms Treated with the Present Invention p Viruses arethe preferred infectious organism treated with the method of the presentinvention. Viral infectious organisms which may be delipidated by thepresent invention to form modified viral particles include, but are notlimited to the lipid-containing viruses of the following genuses:Alphavirus (alphaviruses), Rubivurus (rubella virus), Flavivirus(Flaviviruses), Pestivirus (mucosal disease viruses), (unnamed,hepatitis C virus), Coronavirus, (Coronaviruses) severe acuterespiratory syndrome (SARS), Torovirus, (toroviruses), Arteivirus,(arteriviruses), Paramyxovirus, (Paramyxoviruses), Rubulavirus(rubulavriuses), Morbillivirus (morbillivuruses), Pneumovirinae (thepneumoviruses), Pneumovirus (pneumoviruses), Vesiculovirus(vesiculoviruses), Lyssavirus (lyssaviruses), Ephemerovirus(ephemeroviruses), Cytorhabdovirus (plant rhabdovirus group A),Nucleorhabdovirus (plant rhabdovirus group B), Filovirus (filoviruses),Influenzavirus A, B (influenza A and B viruses), Influenza virus C(influenza C virus), (unnamed, Thogoto-like viruses), Bunyavirus(bunyaviruses), Phlebovirus (phleboviruses), Nairovirus (nairoviruses),Hantavirus (hantaviruses), Tospovirus (tospoviruses), Arenavirus(arenaviruses), unnamed mammalian type B retroviruses, unnamed,mammalian and reptilian type C retroviruses, unnamed, type Dretroviruses, Lentivirus (lentiviruses), Spumavirus (spumaviruses),Orthohepadnavirus (hepadnaviruses of mammals), Avihepadnavirus(hepadnaviruses of birds), Simplexvirus (simplexviruses), Varicellovirus(varicelloviruses), Betaherpesvirinae (the cytomegaloviruses),Cytomegalovirus (cytomegaloviruses), Muromegalovirus (murinecytomegaloviruses), Roseolovirus (human herpes virus 6, 7, 8),Gammaherpesvirinae (the lymphocyte-associated herpes viruses),Lymphocryptovirus (Epstein-Bar-like viruses), Rhadinovirus(saimiri-ateles-like herpes viruses), Orthopoxvirus (orthopoxviruses),Parapoxvirus (parapoxviruses), Avipoxvirus (fowlpox viruses),Capripoxvirus (sheeppoxlike viruses), Leporipoxvirus (myxomaviruses),Suipoxvirus (swine-pox viruses), Molluscipoxvirus (molluscum contagiosumviruses), Yatapoxvirus (yabapox and tanapox viruses), Unnamed, Africanswine fever-like viruses, Iridovirus (small iridescent insect viruses),Ranavirus (front iridoviruses), Lymphocystivirus (lymphocystis virusesof fish), Togaviridae, Flaviviridae, Coronaviridae, Enabdoviridae,Filoviridae, Paramyxoviridae, Orthomyxoviridae, Bunyaviridae,Arenaviridae, Retroviridae, Hepadnaviridae, Herpesviridae, Poxviridae,and any other lipid-containing virus.

These viruses include the following human and animal pathogens: RossRiver virus, fever virus, dengue viruses, Murray Valley encephalitisvirus, tick-borne encephalitis viruses (including European and fareastern tick-borne encephalitis viruses, California encephalitis virus,St. Louis encephalitis virus, sandfly fever virus, human coronaviruses229-E and OC43 and others causing the common cold, upper respiratorytract infection, probably pneumonia and possibly gastroenteritis), humanparainfluenza viruses 1 and 3, mumps virus, human parainfluenza viruses2, 4a and 4b, measles virus, human respiratory syncytial virus, rabiesvirus, Marburg virus, Ebola virus, influenza A viruses and influenza Bviruses, Arenaviruss: lymphocytic choriomeningitis (LCM) virus; Lassavirus, human immunodeficiency viruses 1 and 2, or any otherimmunodeficiency virus, hepatitis B virus, hepatitis C virus, hepatitisG virus, Subfamily: human herpes viruses 1 and 2, herpes virus B,Epstein-Barr virus), (smallpox) virus, cowpox virus, monkeypox virus,molluscum contagiosum virus, yellow fever virus, poliovirus, Norwalkvirus, orf virus, and any other lipid-containing virus.

Methods of Manufacture of the Modified Viral Particle

One of ordinary skill in the art would appreciate that there may bemultiple delipidation processes employed under the scope of thisinvention. In a preferred embodiment, a solvent system together appliedenergy, for example a mechanical mixing system, is used to substantiallydelipidate the viral particle. The delipidation process is dependentupon the total amount of solvent and energy input into a system. Varioussolvent levels and mixing methods, as described below, may be useddepending upon the overall framework of the process. Although a singlesolvent or multiple solvents may be used for delipidation of virus, itis to be understood that a single solvent is preferred since there isless probability of destroying and denaturing the viral particle.

Exemplary Solvent Systems for Use in Removal of Lipid from Viruses andEffective in Maintaining Integrity of the Viral Particle

The solvent or combinations of solvents to be employed in the process ofpartially or completely delipidating lipid-containing organisms may beany solvent or combination thereof effective in solubilizing lipids inthe viral envelope while retaining the structural integrity of themodified viral particle, which can be measured, in one embodiment, viaprotein recovery. A delipidation process falling within the scope of thepresent invention uses an optimal combination of energy input andsolvent to delipidate the viral particle, while still keeping it intact.Suitable solvents comprise hydrocarbons, ethers, alcohols, phenols,esters, halohydrocarbons, halocarbons, amines, and mixtures thereof.Aromatic, aliphatic, or alicyclic hydrocarbons may also be used. Othersuitable solvents, which may be used with the present invention, includeamines and mixtures of amines. One solvent system is DIPE, eitherconcentrated or diluted in water or a buffer such as a physiologicallyacceptable buffer. One solvent combination comprises alcohols andethers. Another solvent comprises ether or combinations of ethers,either in the form of symmetrical ethers, asymmetrical ethers orhalogenated ethers.

The optimal solvent systems are those that accomplish two objectives:first, at least partially delipidating the infectious organism or viralparticle and second, employing a set of conditions such that there arefew or no deleterious effects on the other plasma proteins. In addition,the solvent system should maintain the integrity of the viral particlesuch that it can be used to initiate an immune response in the patient.It should therefore be noted that certain solvents, solventcombinations, and solvent concentrations may be too harsh to use in thepresent invention because they result in a chemical kill.

It is preferred that the solvent or combination of solvents has arelatively low boiling point to facilitate removal through a vacuum andpossibly heat without destroying the antigenic core of the viralparticle. It is also preferred that the solvent or combination ofsolvents be employed at a low temperature because heat has deleteriouseffects on the proteins contained in biological fluids such as plasma.It is also preferred that the solvent or combination of solvents atleast partially delipidate the viral particle.

Liquid hydrocarbons dissolve compounds of low polarity such as thelipids found in the viral envelopes of the infectious organisms.Particularly effective in disrupting the lipid membrane of a viralparticle are hydrocarbons which are substantially water immiscible andliquid at about 37° C. Suitable hydrocarbons include, but are notlimited to the following: C₅ to C₂₀ aliphatic hydrocarbons such aspetroleum ether, hexane, heptane, octane; haloaliphatic hydrocarbonssuch as chloroform, 1,1,2-trichloro-1,2,2-trifluoroethane,1,1,1-trichloroethane, trichloroethylene, tetrachloroethylene,dichloromethane and carbon tetrachloride; thioaliphatic hydrocarbonseach of which may be linear, branched or cyclic, saturated orunsaturated; aromatic hydrocarbons such as benzene; alkylarenes such astoluene; haloarenes; haloalkylarenes; and thioarenes. Other suitablesolvents may also include saturated or unsaturated heterocycliccompounds such as pyridine and aliphatic, thio- or halo-derivativesthereof.

Suitable esters for use in the present invention include, but are notlimited to, ethyl acetate, propylacetate, butylacetate andethylpropionate. Suitable detergents/surfactants that may be usedinclude but are not limited to the following: sulfates, sulfonates,phosphates (including phospholipids), carboxylates, and sulfosuccinates.Some anionic amphiphilic materials useful with the present inventioninclude but are not limited to the following: sodium dodecyl sulfate(SDS), sodium decyl sulfate, bis-(2-ethylhexyl) sodium sulfosuccinate(AOT), cholesterol sulfate and sodium laurate.

Solvents may be removed from delipidated viral mixtures through the useof additional solvents. For example, demulsifying agents such as ethersmay be used to remove a first solvent such as an alcohol from anemulsion. Removal of solvents may also be accomplished through othermethods, which do not employ additional solvents, including but notlimited to the use of charcoal. Charcoal may be used in a slurry oralternatively, in a column to which a mixture is applied. Pervaporationmay also be employed to remove one or more solvents from delipidatedviral mixtures.

Examples of suitable amines for use in removal of lipid fromlipid-containing organisms in the present invention are those which aresubstantially immiscible in water. Typical amines are aliphaticamines—those having a carbon chain of at least 6 carbon atoms. Anon-limiting example of such an amine is C₆H₁₃NH₂.

Ether is a preferred solvent for use in the method of the presentinvention. Particularly preferred are the C₄-C₈ containing-ethers,including but not limited to ethyl ether, diethyl ether, and propylethers (including but not limited to di-isopropyl ether). Asymmetricalethers may also be employed. Halogenated symmetrical and asymmetricalethers may also be employed.

Low concentrations of ethers may be employed to remove lipids when usedalone and not in combination with other solvents. For example, a lowconcentration range of ethers include 0.5% to 30%. Such concentrationsof ethers that may be employed include, but are not limited to thefollowing: 0.625%, 1.0% 1.25%, 2.5%, 5.0% and 10% or higher. It has beenobserved that dilute solutions of ethers are effective. Such solutionsmay be aqueous solutions or solutions in aqueous buffers, such asphosphate buffered saline (PBS). Other physiological buffers may beused, including but not limited to bicarbonate, citrate, Tris,Tris/EDTA, and Trizma. Preferred ethers are di-isopropyl ether (DIPE)and diethyl ether (DEE).

When used in the present invention, appropriate alcohols are those whichare not appreciably miscible with plasma or other biological fluids.Such alcohols include, but are not limited to, straight chain andbranched chain alcohols, including pentanols, hexanols, heptanols,octanols and those alcohols containing higher numbers of carbons.

When alcohols are used in combination with another solvent, for example,an ether, a hydrocarbon, an amine, or a combination thereof, C₁-C₈containing alcohols may be used. Alcohols for use in combination withanother solvent include C₄-C₈ containing alcohols. Accordingly, alcoholsthat fall within the scope of the present invention are butanols,pentanols, hexanols, heptanols and octanols, and iso forms thereof, inparticular, C₄ alcohols or butanols (1-butanol and 2-butanol). Thespecific alcohol choice is dependent on the second solvent employed.

Ethers and alcohols can be used in combination as a first solvent fortreating the fluid containing the lipid-containing virus, or viralparticle. Any combination of alcohol and ether may be used provided thecombination is effective to at least partially remove lipid from theinfectious organism, without having deleterious effects on the plasmaproteins. In one embodiment, lipid is removed from the viral envelope ofthe infectious organism. When alcohols and ether are combined as a firstsolvent for treating the infectious organism contained in a fluid,ratios of alcohol to ether in this solvent are about 0.01%-60% alcoholto about 40%-99.99% of ether, with a specific ratio of about 10%-50% ofalcohol with about 50%-90% of ether, with a more specific ratio of about20%-45% alcohol and about 55%-80% ether.

One combination of alcohol and ether is the combination of butanol anddi-isopropyl ether (DIPE). When butanol and DIPE are combined as a firstsolvent for treating the infectious organism contained in a fluid,ratios of butanol to DIPE in this solvent are about 0.01%-60% butanol toabout 40%-99.99% of DIPE, with a specific ratio of about 10%-50% ofbutanol with about 50%-90% of DIPE, with a more specific ratio of about20%-45% butanol and about 55%-80% DIPE.

Another combination of alcohol and ether is the combination of butanolwith diethyl ether (DEE). When butanol is used in combination with DEEas a first solvent, ratios of butanol to DEE are about 0.01%-60% butanolto about 40%-99.99% of DEE, with a more specific ratio of about 10%-50%of butanol with about 50%-90% of DEE, with a most specific ratio ofabout 20%-45% butanol and about 55%-80% DEE. One specific ratio ofbutanol and DEE in a first solvent is about 40% butanol and about 60%DEE. This combination of about 40% butanol and about 60% DEE (vol:vol)has been shown to have no significant effect on a variety of biochemicaland hematological blood parameters, as shown for example in U.S. Pat.No. 4,895,558.

Biological Fluids and Treatment thereof for Reducing Infectivity ofInfectious, Lipid-Containing Organisms

As stated above, various biological fluids may be treated with themethod of the present invention in order to reduce the levels ofinfectivity of the lipid-containing organism in the biological fluid andto create modified viral particles. In a preferred embodiment, plasmaobtained from an animal or human is treated with the method of thepresent invention in order to reduce the concentration and/orinfectivity of lipid-containing infectious organisms within the plasmaand to create modified viral particles. In this embodiment, plasma maybe obtained from an animal or human patient by withdrawing blood fromthe patient using well-known methods and treating the blood in order toseparate the cellular components of the blood (red and white cells) fromthe plasma. Such methods for treating the blood are known to one ofordinary skill in the art and include but are not limited tocentrifugation and filtration. One of ordinary skill in the artunderstands the proper centrifugation conditions for separating suchlipid-containing organisms from the red and white cells. Filtration mayinclude diafiltration or filtration through membranes with pore sizesthat separate the lipid-containing organism, such as the cell-freevirus, from the red and white cells. Use of the present inventionpermits treatment of lipid-containing organisms, for example those foundwithin plasma, without having deleterious effects on other plasmaproteins and maintaining the integrity of the viral core.

Viruses are typically retained in the plasma and are affected by thetreatment of the plasma with the method of the present invention. Incases where the lipid-containing organism to be treated is substantiallylarger, and may pellet with red and white blood cells under typicalcentrifugation conditions for separating cells from plasma, thelipid-containing organism may be separated from the red and white cellsusing techniques known to one of ordinary skill in the art.

Treatment of lipid-containing organisms in biological fluids other thanblood and plasma does not generally involve separation of the cells fromthe fluid prior to initiation of the delipidation procedure. Forexample, follicular fluid and peritoneal fluid may be treated with thepresent invention to affect the levels and infectivity oflipid-containing organisms without deleterious effects on proteincomponents. The treated fluid may subsequently be reintroduced into theanimal or human from which it was obtained. Treatment of these non-bloodtypes of fluids affects the lipid-containing organisms in the fluid,such as the virus.

Once a biological fluid, such as plasma, is obtained either in thismanner, or for example, from a storage facility housing bags of plasma,the plasma is contacted with a first organic solvent, as describedabove, capable of solubilizing lipid in the lipid-containing infectiousorganism. The first organic solvent is combined with the plasma in aratio wherein the first solvent is present in an amount effective tosubstantially solubilize the lipid in the infectious organism, forexample, dissolve the lipid envelope that surrounds the virus. Exemplaryratios of first solvent to plasma (expressed as a ratio of first organicsolvent to plasma) are described in the following ranges:0.5-4.0:0.5-4.0; 0.8-3.0:0.8-3.0; and 1-2:0.8-1.5. Various other ratiosmay be applied, depending on the nature of the biological fluid. Forexample, in the case of cell culture fluid, the following ranges may beemployed of first organic solvent to cell culture fluid:0.5-4.0:0.5-4.0; 0.8-3.0:0.8-3.0; and 1-2:0.8-1.5.

After contacting the fluid containing the infectious organism with thefirst solvent as described above, the first solvent and fluid are mixed,using methods including but not limited to one of the following suitablemixing methods: gentle stirring; vigorous stirring; vortexing; swirling;homogenization; and end-over-end rotation.

The amount of time required for adequate mixing of the first solventwith the fluid is related to the mixing method employed. Fluids aremixed for a period of time sufficient to permit intimate contact betweenthe organic and aqueous phases, and for the first solvent to at leastpartially or completely solubilize the lipid contained in the infectiousorganism. Typically, mixing will occur for a period of about 10 secondsto about 24 hours, possibly about 10 seconds to about 2 hours, possiblyapproximately 10 seconds to approximately 10 minutes, or possibly about30 seconds to about 1 hour, depending on the mixing method employed.Non-limiting examples of mixing durations associated with differentmethods include 1) gentle stirring and end-over-end rotation for aperiod of about 10 seconds to about 24 hours, 2) vigorous stirring andvortexing for a period of about 10 seconds to about 30 minutes, 3)swirling for a period of about 10 seconds to about 2 hours, or 4)homogenization for a period of about 10 seconds to about 10 minutes.

Separation of Solvents

After mixing of the first solvent with the fluid, the solvent isseparated from the fluid being treated. The organic and aqueous phasesmay be separated by any suitable manner known to one of ordinary skillin the art. Since the first solvent is typically immiscible in theaqueous fluid, the two layers are permitted to separate and theundesired layer is removed. The undesired layer is the solvent layercontaining dissolved lipids and its identification, as known to one ofordinary skill in the art, depends on whether the solvent is more orless dense than the aqueous phase. An advantage of separation in thismanner is that dissolved lipids in the solvent layer may be removed.

In addition, separation may be achieved through means, including but notlimited to the following: removing the undesired layer via pipetting;centrifugation followed by removal of the layer to be separated;creating a path or hole in the bottom of the tube containing the layersand permitting the lower layer to pass through; utilization of acontainer with valves or ports located at specific lengths along thelong axis of the container to facilitate access to and removal ofspecific layers; and any other means known to one of ordinary skill inthe art. Another method of separating the layers, especially when thesolvent layer is volatile, is through distillation under reducedpressure or evaporation at room temperature, optionally combined withmild heating. In one embodiment employing centrifugation, relatively lowg forces are employed, such as 900×g for about 5 to 15 minutes toseparate the phases.

Another method of separating solvent is through the used of charcoal,preferably activated charcoal. This charcoal is optionally contained ina column. Alternatively the charcoal may be used in slurry form. Variousbiocompatible forms of charcoal may be used in these columns.Pervaporation methods and use of charcoal to remove solvents arepreferred methods for removing solvent.

Following separation of the first solvent from the treated fluid, someof the first solvent may remain entrapped in the aqueous layer as anemulsion. Optionally, a de-emulsifying agent is employed to facilitateremoval of the trapped first solvent. The de-emulsifying agent may beany agent effective to facilitate removal of the first solvent. Apreferred de-emulsifying agent is ether and a more preferredde-emulsifying agent is diethyl ether. The de-emulsifying agent may beadded to the fluid or in the alternative the fluid may be dispersed inthe de-emulsifying agent. In vaccine preparation, alkanes in a ratio ofabout 0.5 to 4.0 to about 1 part of emulsion (vol:vol) may be employedas a de-emulsifying agent, followed by washing to remove the residualalkane from the remaining delipidated organism used for preparing thevaccine. Preferred alkanes include, but are not limited to, pentane,hexane and higher order straight and branched chain alkanes.

The de-emulsifying agent, such as ether, may be removed through meansknown to one of skill in the art, including such means as described inthe previous paragraph. One convenient method to remove thede-emulsifying agent, such as ether, from the system, is to permit theether to evaporate from the system in a running fume hood or othersuitable device for collecting and removing the de-emulsifying agentfrom the environment. In addition, de-emulsifying agents may be removedthrough application of higher temperatures, for example from about 24 to37° C. with or without pressures of about 10 to 20 mbar. Another methodto remove the de-emulsifying agent involves separation bycentrifugation, followed by removal of organic solvent throughaspiration, further followed by evaporation under reduced pressure (forexample 50 mbar) or further supply of an inert gas, such as nitrogen,over the meniscus to aid in evaporation. Yet another method of removinga first solvent or a demulsifying agent is through the use ofadsorbants, such as charcoal. The charcoal is preferably activatedcharcoal. This charcoal is optionally contained in a column, asdescribed above. Still another method of removing solvent is the use ofhollow fiber contactors. Pervaporation methods and charcoal adsorbantmethods of removing solvents are preferred.

Methods of Treating Biological Fluids (Delipidation)

It is to be understood that the method of the present invention may beemployed in either a continuous or discontinuous manner. That is, in acontinuous manner, a fluid may be fed to a system employing a firstsolvent which is then mixed with the fluid, separated, and optionallyfurther removed through application of a de-emulsifying agent. Thecontinuous method also facilitates subsequent return of the fluidcontaining delipidated infectious organism to a desired location. Suchlocations may be containers for receipt and/or storage of such treatedfluid, and may also include the vascular system of a human or animal orsome other body compartment of a human or animal, such as the pleural,pericardial, peritoneal, and abdominopelvic spaces.

In one embodiment of the continuous method of the present invention, abiological fluid, for example, blood, is removed from an animal or ahuman through means known to one of ordinary skill in the art, such as acatheter. Appropriate anti-clotting factors as known to one of ordinaryskill in the art are employed, such as heparin,ethylenediaminetetraacetic acid (EDTA) or citrate. This blood is thenseparated into its cellular and plasma components through the use of acentrifuge. The plasma is then contacted with the first solvent andmixed with the first solvent to effectuate lipid removal from theinfectious organism contained within the plasma. Following separation ofthe first solvent from the treated plasma, a de-emulsifying agent isoptionally employed to remove entrapped first solvent. After ensuringthat acceptable levels (non-toxic) of first solvent or de-emulsifyingagent, if employed, are found within the plasma containing thedelipidated infectious organism, the plasma is then optionally combinedwith the cells previously separated from the blood to form a new bloodsample containing at least partially delipidated viral particles, alsocalled modified viral particles herein.

Through the practice of this method, the infectivity of the infectiousorganism is greatly reduced or eliminated. Following recombination withthe cells originally separated from the blood, this sample may bereintroduced into either the vascular system or some other system of thehuman or animal. The effect of such treatment of plasma removed from thehuman or animal and return of the sample containing the partially orcompletely delipidated infectious organism, or modified viral particle,to the human or animal causes a net decrease in the concentration andinfectivity of the infectious organism contained within the vascularsystem of the human or animal. In addition to decreasing theconcentration and infectivity of the infectious organism containedwithin the vascular system, the modified viral particle serves toinitiate an autologous immune response in the patient. In this manner,infectious viral load is reduced. In this mode of operation, the methodof the present invention is employed to treat body fluids in acontinuous manner—while the human or animal is connected to anextracorporeal device for such treatment.

In yet another embodiment, the discontinuous or batch mode, the human oranimal is not connected to an extracorporeal device for processingbodily fluids with the method of the present invention. In adiscontinuous mode of operation, the present invention employs a fluidpreviously obtained from a human or animal, which may include, but isnot limited to plasma, lymphatic fluid, or follicular fluid. The samplemay be contained within a blood bank or in the alternative, drawn from ahuman or animal prior to application of the method. The sample may be areproductive fluid or any fluid used in the process of artificialinsemination or in vitro fertilization. The sample may also be one notdirectly obtained from a human or animal but rather any fluid containinga potentially infectious organism, such as cell culture fluid. In thismode of operation, this sample is treated with the method of the presentinvention to produce a new sample which contains at least partially orcompletely delipidated infectious organisms, or modified viralparticles. One embodiment of this mode of the present invention is totreat plasma samples previously obtained from animals or humans andstored in a blood bank for subsequent transfusion. These samples may betreated with the method of the present invention to minimize oreliminate transmission of infectious disease, such as HIV, hepatitis,cytomegalovirus, from the biological sample.

Delipidation of an infectious organism can be achieved by various means.A batch method can be used for fresh or stored biological fluids, forexample, fresh frozen plasma. In this case a variety of the describedorganic solvents or mixtures thereof can be used for viral inactivation.Extraction time depends on the solvent or mixture thereof and the mixingprocedure employed.

Kits

The kits of the present invention generally comprise containers used fordifferent purposes and are depicted in FIGS. 1-3. A first container 10generally contains one or more first extraction solvents 20. This firstcontainer 10 contains means 15 for removing the first extraction solventfrom the opening 70 of the container 10. Such means may be a componentof the first container 10 or a separate component adapted to functionwith the first container 10. Such means include, but are not limited to,any type of cap 15, spout, funnel, penetrable seal, penetrablediaphragm, tube 60, pipette, or other means for removing the one or morefirst extraction solvents 20 or for introducing a fluid 30 containinglipid-containing virus into the first container 10.

A second container 50 contains the fluid 30 containing lipid-containingvirus to be delipidated.

In one embodiment, a third container 70 is used for mixing the fluid 30containing lipid-containing virus to be delipidated and the firstextraction solvent 20. Mixing can occur through agitation, inversion,shaking, or other means to agitate the third container 70 to a degreesufficient to mix the fluid 30 and the first extraction solvent 20 toform a mixture 72. After the mixing step, the first extraction solventcontaining the dissolved lipids from the fluid or from the virusesseparates from the fluid. At this point, the delipidated fluid may beremoved through any means 75 such as pouring, decanting, pipetting,applying a vacuum connected to a tube or pipette, or any other meansknown to one of ordinary skill in the art of removing separated fluids.

A fourth container 80 optionally receives the delipidated fluid andmodified viral particles 82 originating from the third container 70.Alternatively, the delipidated fluid containing the modified viralparticles is administered into the patient through a tube, catheter, anintravenous line, an intraarterial line or other means withoutintroduction into a fourth container 80.

In one embodiment, the first container 10 containing the firstextraction solvent 20, has sufficient additional volume within it toreceive the fluid 30 containing lipid-containing virus to bedelipidated. In this embodiment, mixing of the first extraction solvent20 and the fluid containing lipid-containing virus 30 to be delipidatedoccurs within the first container 10. In this embodiment, a separatecontainer for mixing the fluid 30 and the first extraction solvent 20,referred to as the third container 70, is not required. After mixingoccurs, the first extraction solvent containing the dissolved lipidsseparates from the delipidated fluid and the modified viral particles.At this point, the delipidated fluid and the modified viral particlesmay be introduced into another container, analogous to the fourthcontainer described above, for subsequent introduction into a patient orfor optional additional extraction of the first extraction solvent witha second extraction solvent 92.

In another embodiment, when a second extraction solvent 92 is optionallyemployed to assist in removal of the first extraction solvent 20, afifth container 90 is included which contains the second extractionsolvent 92. This second extraction solvent 92 may be added to themixture 72 described above in the third container 70, mixed and thenpermitted to separate from the delipidated fluid and modified viralparticles. Alternatively, the second extraction solvent 92 may be addedto the fourth container 80 described above, mixed and then permitted toseparate from the delipidated fluid and modified viral particles ifadditional removal of residual first extraction solvent is desired. Inyet another alternative embodiment, the second extraction solvent 92 maybe added to the first container 10 described above containing themixture of the fluid 30 containing the lipid containing virus and thefirst extraction solvent 20, mixed, and then permitted to separate fromthe delipidated fluid and modified viral particles if mixing of thefluid 30 containing the lipid containing virus and the first extractionsolvent 20, separation and additional extraction of the first extractionsolvent 20 using a second extraction solvent 92 are all performed in thefirst container 10.

The containers described above may be graduated for easy determinationof volume within a container.

Optionally, means for removing or introducing a biological fluid from apatient comprising a venipuncture system may be included in a kit. Suchsystems are well known to those skilled in the art of removing orreplacing fluids, for example vascular fluids, including withoutlimitation a vac tube, hypodermic syringe connected to an intravascularneedle 62, 112, tubing 60, 110 or an intravascular needle 62, 112,connected through tubing 60, 110 to a bag for collection of blood. Anyof these devices may be optionally incorporated into the kit. A sensormay be added to the kit for determining the level of a first extractionsolvent and optionally a second extraction solvent in the delipidatedfluid.

Suitable materials for use in any of the apparatus components asdescribed herein include materials that are biocompatible, approved formedical applications that involve contact with internal body fluids, andin compliance with U.S. PV1 or ISO 10993 standards. Further, thematerials should not substantially degrade during at least a single use,from for instance, exposure to the solvents used in the presentinvention. The materials should typically be sterilizable, preferably byradiation or ethylene oxide (EtO) sterilization. Such suitable materialsshould be capable of being formed into objects using conventionalprocesses, such as, but not limited to, extrusion, injection molding andothers. Materials meeting these requirements include, but are notlimited to, nylon, polypropylene, polycarbonate, acrylic, polysulphone,polyvinylidene fluoride (PVDF), fluoroelastomers such as VITON,available from DuPont Dow Elastomers L.L.C., thermoplastic elastomerssuch as SANTOPRENE, available from Monsanto, polyurethane, polyvinylchloride (PVC), polytetrafluoroethylene (PTFE), polyphenylene ether(PFE), perfluoroalkoxy copolymer (PFA), which is available as TEFLON PFAfrom E.I. du Pont de Nemours and Company, and combinations thereof.

Through the use of the kits and methods of the present invention, levelsof lipid in lipid-containing viruses in a fluid are reduced, and thefluid, for example, delipidated plasma containing the modified viralparticles may be administered to the patient. Such fluid containsmodified viral particles that are not infective. These modified viralparticles induce an immune response in the recipient to epitopes on themodified viral particles. Alternatively the modified viral particles maybe further isolated from the delipidated fluid and combined with apharmaceutically acceptable carrier, and optionally an adjuvant andadministered as a vaccine composition to a human or an animal to inducean immune response in the recipient.

Vaccine Production

The modified viral particle, which is at least partially orsubstantially delipidated and has immunogenic properties is combinedwith a pharmaceutically acceptable carrier to make a compositioncomprising a vaccine. This vaccine composition is optionally combinedwith an adjuvant or an immunostimulant and administered to an animal ora human. It is to be understood that vaccine compositions may containmore than one type of modified viral particle or component thereof, inorder to provide protection against more than one disease aftervaccination. Such combinations may be selected according to the desiredimmunity. For example, preferred combinations may be, but are notlimited to HIV and hepatitis or influenza and hepatitis. Morespecifically, the vaccine can comprise a plurality of modified viralparticles having patient-specific antigens and modified viral particleshaving non-patient specific antigens or stock viral particles that haveundergone the delipidation process of the present invention.

The remaining particles of the organism are retained in the delipidatedbiological fluid, and when reintroduced into the animal or human, arepresumably ingested by phagocytes. The number of viral particlesisolated and modified by the delipidation treatment is determined bycounting the particles before and after treatment.

Administration of Vaccine Produced With the Method of the PresentInvention

When a delipidated infectious organism, for example one in the form of amodified viral particle with exposed antigenic determinants, isadministered to an animal or a human, it is typically combined with apharmaceutically acceptable carrier to produce a vaccine, and optionallycombined with an adjuvant or an immunostimulant as known to one ofordinary skill in the art. The vaccine formulations may conveniently bepresented in unit dosage form and may be prepared by conventionalpharmaceutical techniques known to one of ordinary skill in the art.Such techniques include uniformly and intimately bringing intoassociation the active ingredient and the liquid carriers(pharmaceutical carrier(s) or excipient(s)). Formulations suitable forparenteral administration include aqueous and non-aqueous sterileinjection solutions which may contain anti-oxidants, buffers,bacteriostats and solutes which render the formulation isotonic with theblood of the intended recipient; and aqueous and non-aqueous sterilesuspensions which may include suspending agents and thickening agents.

The formulations may be presented in unit-dose or multi-dosecontainers—for example, sealed ampules and vials—and may be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid carrier, for example, water for injections, immediatelyprior to use. The vaccine may be stored at temperatures of from about 4°C. to −100° C. The vaccine may also be stored in a lyophilized state atdifferent temperatures including room temperature. Extemporaneousinjection solutions and suspensions may be prepared from sterilepowders, granules and tablets commonly used by one of ordinary skill inthe art. The vaccine may be sterilized through conventional means knownto one of ordinary skill in the art. Such means include, but are notlimited to filtration, radiation and heat. The vaccine of the presentinvention may also be combined with bacteriostatic agents, such asthimerosal, to inhibit bacterial growth.

Preferred unit dosage formulations are those containing a dose or unit,or an appropriate fraction thereof, of the administered ingredient. Itshould be understood that in addition to the ingredients, particularlymentioned above, the formulations of the present invention may includeother agents commonly used by one of ordinary skill in the art.

The vaccine may be administered through different routes, such as oral,including buccal and sublingual, rectal, parenteral, aerosol, nasal,intramuscular, subcutaneous, intradermal, intravenous, intraperitoneal,and topical.

The vaccine of the present invention may be administered in differentforms, including but not limited to solutions, emulsions andsuspensions, microspheres, particles, microparticles, nanoparticles, andliposomes. It is expected that from about 1 to 5 dosages may be requiredper immunization regimen. One of ordinary skill in the medical orveterinary arts of administering vaccines will be familiar with theamount of vaccine to be administered in an initial injection and inbooster injections, if required, taking into consideration, for example,the age and size of an a patient.

Vaccination Schedule

The vaccines of the present invention may be administered before, duringor after an infection. The vaccine of the present invention may beadministered to either humans or animals. In one embodiment, the viralload (one or more viruses) of a human or an animal may be reduced bydelipidation treatment of the plasma. The same individual may receive avaccine directed to the one or more viruses, thereby stimulating theimmune system to combat against the virus that remains in theindividual. The optimal time for administration of the vaccine is aboutone to three months before the initial infection. However, the vaccinemay also be administered after initial infection to ameliorate diseaseprogression or to treat the disease.

Adjuvants

A variety of adjuvants known to one of ordinary skill in the art may beadministered in conjunction with the modified viral particles in thevaccine composition. Such adjuvants include, but are not limited to thefollowing: polymers, co-polymers such aspolyoxyethylene-polyoxypropylene co-polymers, including blockco-polymers; polymer P1005; monotide ISA72; Freund's complete adjuvant(for animals); Freund's incomplete adjuvant; sorbitan monooleate;squalene; CRL-8300 adjuvant; alum; QS 21, muramyl dipeptide; trehalose;bacterial extracts, including mycobacterial extracts; detoxifiedendotoxins; membrane lipids; water-in-oil mixtures,water-in-oil-in-water mixtures or combinations thereof.

Suspending Fluids and Carriers

A variety of suspending fluids or carriers known to one of ordinaryskill in the art may be employed to suspend the vaccine composition.Such fluids include without limitation: sterile water, saline, buffer,or complex fluids derived from growth medium or other biological fluids.Preservatives, stabilizers and antibiotics known to one of ordinaryskill in the art may be employed in the vaccine composition.

The following experimental examples are illustrative in showing that adelipidation process of the viral particle occurred and in particular,that the viral particle was modified and noted to exhibit a positiveimmunogenic response in the species from which it was derived. It willbe appreciated that other embodiments and uses will be apparent to thoseskilled in the art and that the invention is not limited to thesespecific illustrative examples or preferred embodiments.

EXAMPLE 1

A. Delipidation of Serum Produces Duck Hepatitis B Virus (DHBV) havingReduced Infectivity

A standard duck serum pool (Camden) containing 10⁶ ID₅₀ doses of DHBVwas used. ID₅₀ is known to one of ordinary skill in the art as theinfective dosage (ID) effective to infect 50% of animals treated withthe dose. Twenty-one ducklings were obtained from a DHBV negative flockon day of hatch. These ducklings were tested at purchase and shown to beDHBV DNA negative by dot-blot hybridization.

The organic solvent system was mixed in the ratio of 40% butanol to 60%diisopropyl ether. The mixed organic solvent system (4 ml) was mixedwith the standard serum pool (2 ml) and gently rotated for 1 hour atroom temperature. The mixture was centrifuged at 400×g for 10 minutesand the lower aqueous phase (containing the plasma) removed at roomtemperature. The aqueous phase was then mixed with an equal volume ofdiethyl ether and centrifuged as before to remove any remaininglipid/solvent mixture. The aqueous phase was again removed and mixedwith an equal volume of diethyl ether and re-centrifuged. The aqueousphase was removed and any residual diethyl ether was removed by airingin a fume cabinet at room temperature for about 1 hour. The delipidatedplasma, with or without viral particles was stored at −20° C.

The positive and negative control duck sera were diluted in phosphatebuffered saline (PBS). Positive controls: 2 ml of pooled serumcontaining 10⁶ID₅₀ doses of DHBV was mixed with 4 ml of PBS. Negativecontrols: 2 ml of pooled DHBV negative serum was mixed with 4 ml of PBS.Residual infectivity was tested by inoculation of 100 μl of either testsample (n=7), negative (n=7) or positive (n=7) controls into theperitoneal cavities of day-old ducks. Controls were run with DHBVnegative serum treated with organic solvents and subsequently mixed withPBS and injected into recipient ducks.

One of the positive control ducks died between 4 and 6 days of age andwas excluded from further analysis. A further 3 positive control ducksdied between 9 and 10 days of age, and two treatment and one negativecontrol died on day 11. It was decided to terminate the experiment. Theremaining ducklings were euthanized on day 12 with sodiumpentibarbitone, i.v., and their livers removed for DHBV DNA analysis asdescribed by Deva et al (J. Hospital Infection 33:119-130, 1996). Allseven negative control ducks remained DHBV negative. Livers of all sixpositive control ducks were DHBV positive. All seven test ducks remainednegative for DHBV DNA in their liver.

Delipidation of serum using the above solvent system resulted in DHBVhaving reduced infectivity. None of the ducklings receiving treatedserum became infected. Although the experiment had to be terminated onday 12 instead of day 14, the remaining positive control ducks werepositive for DHBV (3/3 were DHBV positive by day 10). This suggests thatsufficient time had elapsed for the treated ducks to become DHBVpositive in the liver and that the premature ending of the experimenthad no bearing on the results.

B. Delipidated DHBV Positive Serum as a Vaccine to Prevent DHBVInfection

The efficacy of the delipidation procedure to provide a patient specific“autologous” vaccine against Duck Hepatitis B Virus (DHBV) was examined.Approximately 16 Pekin cross ducklings were obtained from a DHBVnegative flock of ducklings on the day of hatch. The ducklings weretested and determined to be DHBV negative by analysis of DHBV DNA usingdot-blot hybridization. The ducks were divided into the following threegroups:

TABLE 1 # of Ducks Vaccine Administered Results GROUP 1 6 Test Vaccine5/6 ducks remained DHBV negative following challenge GROUP 2 4 ShamVaccine [Glutaraldehyde- 4/4 ducks became DHBV inactivated DHBV(chemical kill)] positive following challenge. GROUP 3 6 Mock Vaccine6/6 ducks became DHBV (Control) [Phosphate Buffered Saline (PBS)]positive following challenge.

1. Glutaraldehyde Inactivation

Glutaraldehyde inactivation was achieved as known by those of ordinaryskill in the art by fixation with a dilute solution of glutaraldehyde atabout 1:250. Glutaraldehyde is a well known cross linking agent.

2. Delipidation Procedure

An organic solvent system was employed to perform delipidation of serum.The solvent system consisted of a ratio of 40% butanol (analyticalreagent grade) and 60% diisopropyl ether and was mixed with the serum ina 2:1 ratio. Accordingly, 4 ml of the organic solvent was mixed with 2ml of the serum and rotated for 1 hour. This mixture was centrifuged atapproximately 400×g for 10 minutes followed by removal of the aqueousphase. The aqueous phase was then mixed with an equal volume of diethylether and centrifuged at 400×g for 10 minutes. Next, the aqueous phasewas removed and mixed with an equal volume of diethyl ether and rotatedend-over-end at 30 rpm for about 1 hour, and centrifuged at 400×g for 10minutes. The aqueous phase was removed and the residual diethyl etherwas removed through evaporation in a fume cabinet for approximately 10to 30 minutes. The treated serum remained following removal of diethylether and was used to produce the vaccine. The delipidation procedurecontrol involved subjecting the DHBV negative serum to the samedelipidation procedure as the DHBV positive serum.

3. Vaccine Production

TABLE 2 First Dose Second Dose Third Dose (injected with 200 μl of(injected with 300 μl (injected with 300 μl of respective vaccine intoof respective vaccine respective vaccine peritoneal cavity on Day 8intramuscularly on intramuscularly on Day 22 Vaccine Type post-hatch)Day 16 post-hatch) post-hatch) TEST A 40 μl aliquot of the A 40 μlaliquot of the A 200 μl aliquot of the delipidated serum was mixeddelipidated serum delipidated serum was mixed with 1960 μl of phosphatewas mixed with with 1800 μl of PBS and then buffered saline (PBS) 1960μl of PBS and emulsified in 1000 μl of Freund's then emulsified inIncomplete Adjuvant. 1000 μl of Freund's Incomplete Adjuvant. SHAM A 200μl aliquot of DHBV A 200 μl aliquot of A 200 μl aliquot of DHBVpositiv$$ (DHBV positive serum pool #4 DHBV positive serum serum pool #4(20.4.99) was SERUM (20.4.99) was mixed with pool #4 (20.4.99) was mixedwith 300 μl of PBS and CONTROL) 300 μl of PBS and 100 μl of a mixed with300 μl of 100 μl 2% glutaraldehyde solution PBS and 100 μl Aidal Plus(Whiteley Chemicals) (Aidal Plus from Whiteley Aidal Plus (Whiteley andincubated for 10 minutes to Chemicals) and incubated for Chemicals) andinactivate the DHBV. A 40 μl 10 minutes to inactivate the incubatedaliquot of the inactivated DHBV. A 40 μl aliquot of the for 10 minutesto serum/PBS mixture was added to inactivated serum/PBS inactivate theDHBV. 1960 μl PBS and emulsified in mixture was added to 1960 μl A 40 μlaliquot of the 1000 μl Freunds Incomplete PBS. inactivated serum/PBSAdjuvant. mixture was added to 1960 μl PBS and emulsified in 1000 μlFreunds Incomplete Adjuvant. MOCK PBS A 2000 μl aliquot of A 2000 μlaliquot of PBS was (DHBV PBS was emulsified emulsified in 1000 μlFreunds NEGATIVE in 1000 μl Freunds Incomplete Adjuvant. CONTROL)Incomplete Adjuvant.

4. Experimental Procedure

Ducks were challenged with 1000 μl of DHBV positive serum (serum pool20.1.97) on day 29, post-hatch. Serum pool 20.1.97 was shown to have1.8×10¹⁰ genome equivalent (gev)/ml by dot-blot hybridization. Onegenome equivalent (gev) is approximately one viral particle. Ducks werebled prior to full vaccination on days 1 and 10, prior to challenge ondays 17 and 23, and post challenge on days 37, 43 and 52. Their serumwas tested for DHBV DNA by dot-blot hybridization as described by Devaet al. (1995). Ducks were euthanized on day 58 and their livers removed,the DNA extracted and tested for the presence of DHBV by dot-blothybridization as described by Deva et al. (1995).

5. Analysis of Results

a. Test Ducks

-   -   i. Five of the 6 test ducks vaccinated with the test vaccine        remained negative for DHBV DNA in the serum and liver following        challenge. One test duck became positive for DHBV following        challenge.

b. Sham Vaccinated Ducks

-   -   i. All 4 of the ducks vaccinated with glutaraldehyde inactivated        serum became DHBV positive following challenge with DHBV.

c. Mock Vaccinated Ducks

i. Five of the 6 mock-vaccinated negative control ducks became DHBVpositive following challenge.

The Chi-square analysis was used to compare differences betweentreatments. Significantly more control ducks (mock vaccinated) becameDHBV positive following challenge than the ducks vaccinated withdelipidated serum p<0.05).

Vaccination of ducklings with delipidated DHBV positive serum using theabove protocol resulted in prevention of DHBV infection followingchallenge with DHBV positive serum in 5 of 6 ducklings. This suggeststhat the delipidated serum vaccine is capable of inducing a positiveimmunogenic response in vaccinated ducks. It is further believed thatthe delipidation process exposed patient-specific antigens that werepreviously unexposed and/or caused a structural change in the viralparticle structure to enable the positive immunogenic response. Incomparison 5 of 6 mock vaccinated and 4 of 4 sham-vaccinated ducksbecame DHBV positive following vaccination suggesting no induction ofimmunity in these ducks due to lack of immune response.

EXAMPLE 2 A. Delipidation of Cattle Pestivirus (Bovine Viral DiarrheaVirus, BVDV), as a Modelfor Hepatitis C

A standard cattle pestivirus isolate (BVDV) was used in theseexperiments. This isolate, “Numerella” BVD virus, was isolated in 1987from a diagnostic specimen submitted from a typical case of ‘MucosalDisease’ on a farm in the Bega district of New South Wales (NSW),Australia. This virus is non-cytopathogenic, and reacts with all 12 of apanel of monoclonal antibodies raised at the Elizabeth MacarthurAgricultural Institute (EMAI), NSW, Australia, as typing reagents.Therefore, this virus represents a ‘standard strain’ of Australian BVDviruses.

The Numerella virus was grown in bovine MDBK cells tested free ofadventitious viral agents, including BVDV. The medium used for viralgrowth contained 10% adult bovine serum derived from EMAI cattle, all ofwhich tested free of BVDV virus and BVDV antibodies. This serumsupplement has been employed for years to exclude the possibility ofadventitious BVDV contamination of test systems, a common failing inlaboratories worldwide that do not take precautions to ensure the testvirus is the only one in the culture system. Using these tested culturesystems ensured high-level replication of the virus and a high yield ofinfectious virus. Titration of the final viral yield after 5 days growthin MDBK cells showed a titer of 10^(6.8) infectious viral particles perml of clarified (centrifuged) culture medium.

1. Treating Infectious BVDV

100 ml of tissue-culture supernatant, containing 10^(6.8) viralparticles/ml, was harvested from a 150 cm² tissue-culture flask. Thesupernatant was clarified by centrifugation (cell debris pelleted at3000 rpm, 10 min, 4° C.) and 10 ml set aside as a positive control foranimal inoculation (non-treated virus). The remaining 90 ml, containing10^(7.75) infectious virus, was treated using the following protocol:180 ml of a solvent mixture butanol:diisopropyl ether (DIPE) (2:1) wasadded to a 500 ml conical flask and mixed by swirling. The mixture wasthen shaken for 60 min at 30 rpm at room temperature on an orbitalshaker. It was then centrifuged for 10 min at 400×g at 4° C., afterwhich the organic solvent phase was removed and discarded. In subsequentsteps, the bottom layer (aqueous phase) was removed from beneath theorganic phase, improving yields considerably.

The aqueous phase, after the butanol:DIPE treatment, was washed fourtimes with an equal volume of fresh diethyl ether (DEE) to remove allcontaminating traces of butanol. After each washing, the contents of theflask was swirled to ensure even mixing of both aqueous and solventphases before centrifugation as above (400×g, 10 min, 4° C.). After fourwashes, the aqueous phase was placed in a sterile beaker covered with asterile tissue fixed to the top of the beaker with a rubber band toprevent contamination and placed in a fume hood running continuouslyovernight (16 hr) to remove all remaining volatile ether residue fromthe inactivated viral preparation. Subsequent culture of the treatedmaterial demonstrated no contamination. The treated viral preparationwas then stored at 4° C. under sterile conditions until inoculation intotissue culture or animals to test for any remaining infectious virus.

2. Testing of Treated BVDV Preparation

a. Tissue-Culture Inoculation

2 ml of the solvent-treated virus preparation, expected to contain about10^(7.1) viral equivalents, was mixed with 8 ml tissue-culture mediumMinimal Eagles Medium (MEM) containing 10% tested-free adult bovineserum and adsorbed for 60 min onto a monolayer of MDBK cells in a 25 cm²tissue-culture flask. As a positive control, 2 ml of non-treated orsubstantially lipid-containing infectious virus (also containing about10^(7.1) viral equivalents) was similarly adsorbed on MDBK cells in a 25cm² tissue-culture flask. After 60 min, the supernatant was removed fromboth flasks and replaced with normal growth medium (+10% ABS). The cellswere then grown for 5 days under standard conditions before the MDBKcells were fixed and stained using a standard immunoperoxidase protocolwith a mixture of 6 BVDV-specific monoclonal antibodies (EMAI panel,reactive with 2 different BVD viral proteins).

There were no infected cells in the monolayer of MDBK cells that wasinoculated with the organic solvent treated virus. In contrast,approximately 90% of the cells in the control flask (that was inoculatedwith non-inactivated BVDV) were positive for virus as shown by heavy,specific, immunoperoxidase staining. These results showed that, under invitro testing conditions, no infectious virus remained in the treated,at least partially delipidated BVDV preparation.

b. Animal Inoculation

An even more sensitive in vivo test is to inoculate naïve (antibodynegative) cattle with the at least partially delipidated viruspreparation. As little as one infectious viral particle injectedsubcutaneously in such animals is considered to be an infectious cowdose, given that entry into cells and replication of the virus isextremely efficient for BVDV. A group of 10 antibody-negative steers(10-12 months of age) were randomly allocated to 3 groups.

The first group of 6 steers was used to test whether BVDV had reducedinfectivity. The same at least partially delipidated preparation of BVDVdescribed above was used in this example. Two steers were inoculatedwith a vaccine having at least partially delipidated viral particles toact as a positive control for the vaccine group. These two positivecontrol animals were run under separate, quarantined conditions toprevent them from infecting other animals when they developed atransient viraemia after infection (normally at 4-7 days after receivinglive BVDV virus). The two remaining steers acted as negative “sentinel”animals to ensure there was no naturally-occurring pestivirustransmission within the vaccinated group of animals. Antibody levelswere measured in all 10 animals using a validated, competitive ELISAdeveloped at EMAI. This test has been independently validated by CSL Ltdand is marketed by IDEXX Scandinavia in Europe.

The six animals in the first group each received a subcutaneousinjection of 4.5 ml of the at least partially delipidated BVDVpreparation, incorporated in a commercial adjuvant. Since each ml of theat least partially delipidated preparation contained 10^(6.8) viralequivalents, the total viral load before the delipidation process was10^(7.4) tissue culture infectious doses (TCID)₅₀. The positive-controlanimals received 5 ml each of the non-delipidated preparation, that is,10^(7.5) TCID₅₀ injected subcutaneously in the same way as for the firstgroup. The remaining two ‘sentinel’ animals were not given any viralantigens, having been grazed with the first group of animals throughoutthe trial to ensure there was no natural pestivirus activity occurringin the group while the trial took place.

There was no antibody development in any of the vaccinated steersreceiving the at least partially delipidated BVD virus preparation untila second dose of vaccine was given. Thus, at 2 and 4 weeks after asingle dose, none of the 6 steers seroconverted showing that there wasno infectious virus left in a total volume of 27 ml of the at leastpartially delipidated virus preparation. This is the equivalent of atotal inactivation of 10^(8.2) TCID₅₀. In contrast, there were highlevels of both anti-E2 antibodies (neutralizing antibodies) and anti-NS3antibodies at both 2 and 4 weeks after inoculation in the two steersreceiving 5 ml each of the viral preparation prior to delipidation. Thisconfirmed the infectious nature of the virus prior to delipidation.These in vivo results confirm the findings of the in vitrotissue-culture test. The two ‘sentinel’ animals remained seronegativethroughout, showing the herd remained free of natural pestivirusinfections.

The panel of monoclonal antibodies used detected host antibodiesdirected against the major envelope glycoprotein (E2), which is aglycoprotein incorporated in the lipid envelope of the intact virus. Thetest systems also detected antibodies directed against thenon-structural protein, NS3 that is made within cells infected by thevirus. This protein has major regulatory roles in viral replication andis not present within the infectious virus. There was no evidence in thevaccinated cattle that infectious virus was present, indicating allinfectious viral particles had been destroyed. All pestiviruses are RNAviruses. Therefore, there was no viral DNA present in the delipidatedpreparation. These results demonstrate the efficacy of the presentmethod to at least partially delipidate virus such that substantially noinfectious virus is found in animals receiving the delipidated virus.

B. Delipidated BVDV Preparation as a Vaccine in Steers

All six steers that had received an initial dose of 4.5 ml of the atleast partially delipidated BVDV preparation described in above inSection A were again injected subcutaneously with a similar dose at 4weeks after the first priming dose. At this time there were no antibodyresponses after the initial dose. It is normal for an animal to reactafter the second dose. Strong secondary immune responses for anti-E2antibody levels (equivalent to serum neutralizing antibodies SNT) wereobserved in 3 of the 6 steers at 2 weeks after the second dose of the atleast partially delipidated virus. This response was more than 70%inhibition in a competitive ELISA. The remaining 3 animals showed weakantibody responses (23-31% inhibition).

In contrast to the anti-E2 antibody responses, only one animal developeda strong anti-NS3 antibody response (93% inhibition) at 2 weeks afterthe second dose of at least partially delipidated BVDV. A second animalhad a weak anti-NS3 response (29% inhibition) and four animals showed noantibody following administration of 2 doses. This was not unexpectedsince similar responses following administration of at least partiallydelipidated BVDV vaccines have been observed previously. The antibodylevels in steers following 2 doses of the at least partially delipidatedBVDV preparation demonstrate its potential as a vaccine since antiE2antibody levels were measurable in all 6 vaccinated steers at 2 weeksafter the second dose.

EXAMPLE 3

Use of Delipidated SIV to Induce or Augment SIV Specific Humoral andCD4+ T Cell Memory Responses in Mice—a Modelfor a New Auto-VaccinationStrategy against Lentiviral Infection

The following studies focused on the simian equivalent of human HIV,termed SIV. The purpose was to utilize delipidated SIVmac251 (anuncloned highly pathogenic isolate of SIV) to carry out studies todetermine the relative immunogenicity of the delipidated virus in mice.The complete nucleotide sequence of an infectious clone of simianimmunodeficiency virus of macaques, SIVmac239, has been determined.Virus produced from this molecular clone causes AIDS in rhesus monkeysin a time frame suitable for laboratory investigation. The proviralgenome including both long terminal repeats is 10,279 base pairs inlength and contains open reading frames for gag, pol, vif, vpr, vpx,tat, rev, and env. The nef gene contains an in-frame premature stopafter the 92nd codon. At the nucleotide level, SIVmac239 is closelyrelated to SIVmac251 (98%) and SIVmac142 (96%). (Regier DA, DesrosiersAnnual Review Immunology. 1990;8:55-78.)

Experiments were performed to determine the minimal dose of delipidatedsimian immunodeficiency virus (SIV) that would produce a readilyrecognizable boosting of the virus specific humoral and/or cellularimmune response in previously primed Balb/c mice. All experiments werecarried out in a BSL3 facility.

The immunogenicity of the delipidated virus preparation was comparedwith an aliquot of the same virus in its native form. The quality (titerof antibody, the conformational and linear epitope specificity of theantibody, the isotype content of the antibody and the function of theantibody) and quantity of antibody induced by immunization of mice withequivalent protein amounts of the non-delipidated and delipidated viruspreparation were ascertained as described below. Total protein from analiquot of wild type virus and total protein recovered followingdelipidation of the same aliquot of virus were determined using standardquantitative protein assay (Biorad, BCA kit assay, Rockford, Illinois).The total protein profile was determined using SDS-PAGE analysis of thewild type virus and the delipidated virus preparation and the relativeepitope preservation was ascertained by Western Blot comparison of wildtype with delipidated virus.

Equivalent protein amounts of the chemically treated wild type and thedelipidated virus were analyzed for their ability to boost virusspecific immune response in groups of mice. The sera from theseimmunized mice were assayed by ELISA and Western Blot analysis forreactivity against native wild type and for comparison the delipidatedvirus preparation. Spleen cells were assayed for their CD4 and CD8 SIVvirus env and gag specific immune response enhancing capacity asoutlined below. Standard statistical analyses were performed for theanalysis of the data.

Four to six week old healthy female Balb/c mice from the Jackson labs,Bar Harbor, Me were purchased and housed in the BSL2/3 mouse housingfacility at Emory University. Twenty Balb/c mice were each immunizedsubcutaneously with 25 ug of protein of 2-2 dithiopyridine-inactivatedSIVmac251 incorporated in an equal volume of Freunds incompleteadjuvant.

A sufficient quantity of SIVmac251 was delipidated to provide the amountneeded for boosting these mice per schedule. Delipidation consisted ofincubating SIVmac251 with 10% DIPE in phosphate buffered saline (PBS).1.0 ml of a 10% DIPE solution in PBS was prepared and mixed on avortexer until it appeared cloudy.

The virus preparation: A 1 ml tube from Advanced BiotechnologiesSIVmac251 was used as seed stock (Sucrose Gradient Purified Virus 1mg/ml). The supplier reported a titer of 10^(6.7) with total protein of1.074 mg/mL (Pierce BCA protein method) and virus particle count of6.95¹⁰/ml (EM). It was confirmed that the virus had a titer of 10^(7.0)using CEMx174, the first time as a rapid assay, and the second time inquadruplicate cultures/dilution. A measurement of p27 in thispreparation revealed a value of 106 ug/ml. Next, 25 μl of the undilutedviral stock was introduced into 0.6 ml clear snap-cap polypropyleneEppendorf tube.1 Then, 2.5 μl of 10% DIPE solution was added into theEppendorf tube containing virus and vortexed for 15 seconds. The tubewas spun (using an Eppendorf 5810R centrifuge) at room temp at 1000×gfor 2 minutes. No bulk solvent was removed. The solvent was removed byvacuum centrifugation (Speedvac Concentrator Model SVC200H) at 2000 rpmwith no heat for 30 minutes. The volume in the tube was adjusted to 25μl with PBS. Total protein recovery was measured using a Pierce BCAprotocol. Gels (12% SDS-PAGE) were employed for specific proteinrecoveries (env protein, pol protein, gp41, p27 and gag protein) andstained with Coomasie Blue and provided semi-quantitative results usingOD. Western blots were run using serum from SIV-infected monkeys tomeasure envelope protein, gp66, gp41, p27, gag, and p6 gag. The viralinfectivity of the preparation was determined using a luciferase assayand CEM-174 cells. The virus titer was 10^(4.5), a 2.5 log reductionfrom that measured in undelipidated stock. This delipidated SIVpreparation appears to retain greater than 90% of the major proteinconstituents of SIVmac251 such as the gag and env proteins.

Next, the immunogenicity of the modified viral preparation wasdetermined in the twenty adult female Balb/c mice described above thatwere each immunized subcutaneously with 25 ug of protein of 2-2dithiopyridine-inactivated SIVmac251. On day 14, groups 3-6 were boostedwith 10 ug to 0.01 ug (based on total protein of stock) of delipidatedvirus in 0.5 ml normal saline. The estimated actual virus proteincontent was equal to 1/10 that of total protein based on the ratio oftotal protein/p27 protein in stock. The mice were injected with thedelipidated vaccine composition as follows:

TABLE 3 Initial Immunization s.c. 2-2 dithiopyridine- Groups (containing4 inactivated Day 14 - Booster mice each) SIVmac251 Injections i.v.GROUP 1 - Control Non-immunized Administered-saline without delipidatedvirus GROUP 2 Immunized Not administered GROUP 3 Immunized 0.5 mlsaline + 10 ug of delipidated virus GROUP 4 Immunized 0.5 ml saline +1.0 ug of delipidated virus GROUP 5 Immunized 0.5 ml saline + 0.1 ug ofdelipidated virus GROUP 6 Immunized 0.5 ml saline + 0.01 ug ofdelipidated virusFour days after the booster injection, the mice were anesthetized andblood was collected via retro-orbital puncture and intra-cardiacpuncture. About 0.5 ml of blood was collected from each mouse, primarilyfrom intra-cardiac puncture. The blood was permitted to clot at roomtemperature. The spleen of each mouse was aseptically removed andtransported to the lab under double bag containment. The clotted bloodfrom each mouse was centrifuged at about 450×g at room temperature, andserum was collected from tube, transferred to a sterile tube, and storedat −70° C. until use. ELISA was performed to determine antibody titersagainst SIV for each serum sample.

SIV ELISA Protocol

Stocks of positive and negative serum and fluids to be tested werefrozen in aliquots to be used on every plate to standardize each run.

Coated Corning Easy-Plates were washed with 100 ul per well ofpoly-1-lysine at a concentration of 10 ug per ml of PBS, pH 7.2-7.4.Plates were covered and incubated overnight at 4° C. Several plates werecoated at one time and stored for subsequent use. Next, excesspolylysine was removed and the plate dried for a few minutes. About 100ul of 2% Triton-X was added to 100 ul of the stock ABI SiVmac251 thesamples sat for 5 minutes. Next, 50 ul of coating buffer of pH 9.6 wasadded. Next, 100 ul of the viral antigen was added to each well of 5plates, which were covered and incubated at 4° C. overnight.

After the overnight incubation, wells were washed 3 times with PBS-T.The wells then received 200 ul per well of 2% nonfat dry milk in PBS forone hour at room temperature to block non-specific binding. Excess fluidwas removed. About 100 ul of test or control serum diluted at 1/100 in10% RPMI 1640 or PBS with 10% calf serum was added to duplicate wellsand incubated for 2 hours at 37° C. Wells were washed 4 times withPBS-T. Next 100 ul of Southern Biotech (from Fisher) alkalinephosphatase anti Mouse IgG ( diluted 1/800 in media or PBS with 10% calfserum) was added and incubated 1 hour at 37° C. Wells were washed 4times with PBS-T.

The BIORAD Alkaline Phosphatase Substrate kit was used to develop areaction product. One substrate tablet was added for each 5 ml of 1×buffer and mixed. Next 100 ul was added per well and evaluated at about5, 10, 15, 30 and then at 1 hour intervals for color development.

Blank readings were obtained from the media controls when the positivecontrol was above 1.500 and the negative control was 0.100 to 0.200 forthe serum. The results were then recorded and the means and the standarddeviations of the negative control, positive control and theexperimental samples were calculated. The negative cutoff value was themean of the negative control plus 0.150.

Immunogenicity Results

The immunogenicity of the delipidated SIV virus preparation in mice wasexamined with an ELISA assay. The mean optical density (O.D.) wasexamined at 405 nm at various dilutions of serum. Table 4 provides theresults of the ELISA test on serum samples.

TABLE 4 Serum No 10 dil. boost ug boost 1 ug boost 0.1 ug boost 0.01 ugboost 1/100  2.541 3.663 3.289 2.846 2.627 1/500  1.035 2.86 2.055 1.4581.257 1/2500  0.449 1.239 0.855 0.601 0.445 1/12500 0.194 0.463 0.3040.229 0.181 1/62500 0.127 0.151 0.153 0.129 0.123  1/312500 0.11 0.1160.108 0.108 0.107Analysis of Responses of Dissociated Spleen Cells Obtained fromImmunized Mice

A single cell suspension of spleen cells was prepared from eachindividual mouse by gently teasing the splenic capsule and passing thecells through a 25 gauge needle. Spleen cells were dissociated into asingle cell suspension in medium (RPMI 1640 supplemented with 100 ug/mlpenicillin, 100 ug/ml streptomycin, 2 mM L-glutamine), washed twice inmedium and subsequently adjusted to 10 million cells/ml. 0.1 ml of thiscell suspension from each mouse was dispensed into each well of a 96well round bottom microtiter plate containing medium. Remaining cellswere cryopreserved. These spleen cell cultures were then assessed forthe ability of CD4+ and CD8+ T cells to synthesize IFN-gamma by standardintracellular cytokine staining (ICC) and flow cytometry.

Two individual wells containing the duplicate cell cultures from anindividual mouse received either a) 0.1 ml of medium containing 2 ug/mlof each of a pool of 9 SIV envelope (SE) peptides (n=14 pools), or b)0.1 ml of medium containing a pool of 7 SIV gag (SG) peptides (n=17pools). Each pool contained 2 ug/ml of 7 peptides each for SIV env andSIV gag. Controls consisted of spleen cell cultures that received mediaalone (background control) or a previously determined optimumconcentration of phorbol myristic acetate (PMA 1 ug/ml)+ionomycin (0.25ug/ml) for maximal IFN-gamma staining (positive control). The SIVenvpeptides (n=49 individual peptides) were mixed in a grid fashion of a7×7 matrix and the SIV gag peptides (n=72 peptides) were mixed in a gridfashion of a 9×8 matrix which permitted identification of individualpeptide specific immune responses. The SIV env and gag peptides weresynthetic 20 mer peptides that overlapped each other by 12 amino acidsand encompassed the entire SIV env and gag sequence. Peptide pools weremade to contain 2.0 ug/ml of each peptide. For each spleen cellpreparation there were 36 wells of culture. The components of the 9pools and 7 pools of env and gag overlapping peptides are describedbelow. Shown are the peptides that compose the pools with theirrespective position within SiVmac239gag (SG) and env (SE).

TABLE 5 Pool arrangement of individual SIVmac239 env peptides. 7 SGPeptide pools SG18 1 2 3 4 5 6 7 SG19 8 9 10 11 12 13 14 SG20 15 16 17G-6* G-5* 20 21 SG21 22 23 24 25 26 27 28 SG22 29 30 31 32 33 34 35 SG2336 37 38 39 40 41 42 SG24 43 44 45 46 47 48 49

TABLE 6 Pool arrangement of individual SIVmac239 env peptides. 9 SEPeptide pools SE9 1 2 3 4 5 6 7 8 SE10 9 10 11 12 13 14 15 16 SE11 17 1819 20 21 22 23 24 SE12 25 26 27 28 29 30 31 32 SE13 33 34 35 36 37 38 3940 SE14 41 42 43 44 45 46 47 48 SE15 49 50 51 52 53 54 55 56 SE16 57 5859 60 61 62 63 64 SE17 65 66 67 68 69 70 71 72

TABLE 7 SIVmac239 gag overlapping peptides for epitope mapping SEQ IDNO: 1 MGVRNSVLSGKKADELEKIRLR SG1   1-22 SEQ ID NO: 2KKADELEKIRLRPNGKKKYMLK SG2  11-32 SEQ ID NO: 3 LRPNGKKKYMLKHVVWAANELDSG3  21-42 SEQ ID NO: 4 LKHVVWAANELDRFGLAESLLE SG4  31-52 SEQ ID NO: 5LDRFGLAESLLENKEGCQKILS SG5  41-62 SEQ ID NO: 6 LENKEGCQKILSVLAPLVPTGSSG6  51-72 SEQ ID NO: 7 LSVLAPLVPTGSENLKSLYNTV SG7  61-82 SEQ ID NO: 8GSENLKSLYNTVCVIWCIHAEE SG8  71-92 SEQ ID NO: 9 TVCVIWCIHAEEKVKHTEEAKQSG9  81- 102 SEQ ID NO: 10 EEKVKHTEEAKQIVQRHLVVET SG10  91- 112 SEQ IDNO: 11 KQIVQRHLVVETGTTETMPKTS SG11 101- 122 SEQ ID NO: 12ETGTTETMPKTSRPTAPSSGRG SG12 111- 132 SEQ ID NO: 13TSRPTAPSSGRGGNYPVQQIGG SG13 121- 142 SEQ ID NO: 14RGGNYPVQQIGGNYVHLPLSPR SG14 131- 152 SEQ ID NO: 15GGNYVHLPLSPRTLNAWVKLIE SG15 141- 162 SEQ ID NO: 16PRTLNAWVKLIEEKKFGAEVVP SG16 151- 172 SEQ ID NO: 17IEEKKFGAEVVPGFQALSEGCT SG17 161- 182 SEQ ID NO: 18VPGFQALSEGCTPYDINQMLNCVGD G-6 171- 195* SEQ ID NO: 19GCTPYDINQMLNCVGDHQAA G-5 180- 199* SEQ ID NO: 20 NCVGDHQAAMQIIRDIINEEAADSG20 191- 213 SEQ ID NO: 21 IIRDIINEEAADWDLQHPQPAP SG21 202- 223 SEQ IDNO: 22 ADWDLQHPQPAPQQGQLREPSG SG22 212- 233 SEQ ID NO: 23APQQGQLREPSGSDIAGTTSSV SG23 222- 243 SEQ ID NO: 24SGSDIAGTTSSVDEQIQWMYRQ SG24 232- 253 SEQ ID NO: 25SVDEQIQWMYRQQNPIPVGNIY SG25 242- 263* (*) SEQ ID NO: 26RQQNPIPVGNIYRRWIQLGLQK SG26 252- 273(*) SEQ ID NO: 27IYRRWIQLGLQKCVRMYNPTNIL SG27 262- 284(*) SEQ ID NO: 28KCVRMYNPTNILDVKQGPKEPF SG28 273- 294 SEQ ID NO: 29ILDVKQGPKEPFQSYVDRFYKS SG29 283- 304 SEQ ID NO: 30PFQSYVDRFYKSLRAEQTDAAV SG30 293- 314 SEQ ID NO: 31KSLRAEQTDAAVKNWMTQTLLI SG31 303- 324 SEQ ID NO: 32AVKNWMTQTLLIQNANPDCKLV SG32 313- 334 SEQ ID NO: 33LIQNANPDCKLVLKGLGVNPTL SG33 323- 344 SEQ ID NO: 34LVLKGLGVNPTLEEMLTACQGV SG34 333- 354 SEQ ID NO: 35TLEEMLTACQGVGGPGQKARLM SG35 343- 364 SEQ ID NO: 36GVGGPGQKARLMAEALKEALAP SG36 353- 374 SEQ ID NO: 37LMAEALKEALAPVPIPFAAAQQ SG37 363- 384 SEQ ID NO: 38APVPIPFAAAQQRGPRKPIKCW SG38 373- 394 SEQ ID NO: 39AQQRGPRKPIKCWNCGKEGHSA SG39 382- 403 SEQ ID NO: 40KCWNCGKEGHSARQCRAPRRQG SG40 392- 413 SEQ ID NO: 41SARQCRAPRRQGCWKCGKMDHV SG41 402- 423 SEQ ID NO: 42RQGCWKCGKMDHVMAKCPDRQAG SG42 411- 433 SEQ ID NO: 43HVMAKCPDRQAGFLGLGPWGKK SG43 422- 443 SEQ ID NO: 44AGFLGLGPWGKKPRNFPMAQVH SG44 432- 453 SEQ ID NO: 45KKPRNFPMAQVHQGLMPTAPPE SG45 442- 463 SEQ ID NO: 46VHQGLMPTAPPEDPAVDLLKNY SG46 452- 473 SEQ ID NO: 47PEDPAVDLLKNYMQLGKQQREK SG47 462- 483 SEQ ID NO: 48NYMQLGKQQREKQRESREKPYK SG48 472- 493 SEQ ID NO: 49EKQRESREKPYKEVTEDLLHLN SG49 482- 503 SEQ ID NO: 50 YKEVTEDLLHLNSLFGGDQSG50 492- 510 *denotes peptides containing defined or (*)semi definedgag epitopes (156-158)

TABLE 8 Overlapping peptides in Env of SIVmac239 (25-mer with 13-meroverlapping) SEQ ID MGCLGNQLLIAILLLSVYGIYCTLY SE1   1-25 NO: 51 SEQ IDLLLSVYGIYCTLYVTVFYGVPAWRN SE2  13-37 NO: 52 SEQ IDYVTVFYGVPAWRNATIPLFCATKNR SE3  25-49 NO: 53 SEQ IDNATIPLFCATKNRDTWGTTQCLPDN SE4  37-61 NO: 54 SEQ IDRDTWGTTQCLPDNGDYSEVALNVTE SE5  49-73 NO: 55 SEQ IDNGDYSEVALNVTESFDAWNNTVTEQ SE6  61-85 NO: 56 SEQ IDESFDAWNNTVTEQAIEDVWQLFETS SE7  73-97 NO: 57 SEQ IDQAIEDVWQLFETSIKPCVKLSPLCI SE8  85-109 NO: 58 SEQ IDSIKPCVKLSPLCITMRCNKSETDRW SE9  97-121 NO: 59 SEQ IDTMRCNKSETDRWGLTKSITTTAST SE10 109-133 NO: 60 SEQ IDWGLTKSITTTASTTSTTASAKVDMV SE11 121-145 NO: 61 SEQ IDTTSTTASAKVDMVNETSSCIAQDNC SE12 133-157 NO: 62 SEQ IDVNETSSCIAQDNCTGLEQEQMISCK SE13 145-169 NO: 63 SEQ IDCTGLEQEQMISCKFNMTGLKRDKKK SE14 157-181 NO: 64 SEQ IDKFNMTGLKRDKKKEYNETWYSADLV SE15 169-193 NO: 65 SEQ IDKEYNETWYSADLVCEQGNNTGNESR SE16 181-205 NO: 66 SEQ IDVCEQGNNTGNESRCYMNHCNTSVIQ SE17 193-217 NO: 67 SEQ IDRCYMNHCNTSVIQESCDKHYWDAIR SE18 205-229 NO: 68 SEQ IDQESCDKHYWDAIRFRYCAPPGYALL SE19 217-241 NO: 69 SEQ IDRFRYCAPPGYALLRCNDTNYSGFMP SE20 229-253 NO: 70 SEQ IDLRCNDTNYSGFMPKCSKVVVSSCTR SE21 241-265 NO: 71 SEQ IDPKCSKVVVSSCTRMMETQTSTWFGF SE22 253-277 NO: 72 SEQ IDRMMETQTSTWFGFNGTRAENRTYIY SE23 265-289 NO: 73 SEQ IDFNGTRAENRTYIYWHGRDNRTIISL SE24 277-301 NO: 74 SEQ IDYWHGRDNRTIISLNKYYNLTMKCRR SE25 289-313 NO: 75 SEQ IDLNKYYNLTMKCRRPGNKTVLPVTIM SE26 301-325 NO: 76 SEQ IDRPGNKTVLPVTIMSGLVFHSQPIND SE27 313-337 NO: 77 SEQ IDMSGLVFHSQPINDRPKQAWCWFGGK SE28 325-349 NO: 78 SEQ IDDRPKQAWCWFGGKWKDAIKEVKQTI SE29 337-361 NO: 79 SEQ IDKWKDAIKEVKQTIVKHPRYTGTNNT SE30 349-373 NO: 80 SEQ IDIVKHPRYTGTNNTDKINLTAPGGGD SE31 361-385 NO: 81 SEQ IDTDKINLTAPGGGDPEVTFMWTNCRG SE32 373-397 NO: 82 SEQ IDDPEVTFMWTNCRGEFLYCKMNWFLN SE33 385-409 NO: 83 SEQ IDGEFLYCKMNWFLNWVEDRNTANQKP SE34 397-421 NO: 84 SEQ IDNWVEDRNTANQKPKEQHKRNYVPCH SE35 409-433 NO: 85 SEQ IDPKEQHKRNYVPCHIRQIINTWHKVG SE36 421-445 NO: 86 SEQ IDHIRQIINTWHKVGKNVYLPPREGDL SE37 433-457 NO: 87 SEQ IDGKNVYLPPREGDLTCNSTVTSLIAN SE38 445-469 NO: 88 SEQ IDLTCNSTVTSLIANIDWIDGNQTNIT SE39 457-481 NO: 89 SEQ IDNIDWIDGNQTNITMSAEVAELYRLE SE40 469-493 NO: 90 SEQ IDTMSAEVAELYRLELGDYKLVEITPI SE41 481-505 NO: 91 SEQ IDELGDYKLVEITPIGLAPTDVKRYTT SE42 493-517 NO: 92 SEQ IDIGLAPTDVKRYTTGGTSRNKRGVFV SE43 505-529 NO: 93 SEQ IDTGGTSRNKRGVFVLGFLGFLATAGS SE44 517-541 NO: 94 SEQ IDVLGFLGFLATAGSAMGAASLTLTAQ SE45 529-553 NO: 95 SEQ IDSAMGAASLTLTAQSRTLLAGIVQQQ SE46 541-565 NO: 96 SEQ IDQSRTLLAGIVQQQQQLLDVVKRQQE SE47 553-577 NO: 97 SEQ IDQQQLLDVVKRQQELLRLTVWGTKNL SE48 565-589 NO: 98 SEQ IDELLRLTVWGTKNLQTRVTAIEKYLK SE49 577-601 NO: 99 SEQ IDLQTRVTAIEKYLKDQAQLNAWGCAF SE50 589-613 NO: 100 SEQ IDKDQAQLNAWGCAFRQVCHTTVPWPN SE51 601-625 NO: 101 SEQ IDFRQVCHTTVPWPNASLTPKWNNETW SE52 613-637 NO: 102 SEQ IDNASLTPKWNNETWQEWERKVDFLEE SE53 625-649 NO: 103 SEQ IDWQEWERKVDFLEENITALLEEAQIQ SE54 637-661 NO: 104 SEQ IDENITALLEEAQIQQEKNMYELQKLN SE55 649-673 NO: 105 SEQ IDQQEKNMYELQKLNSWDVFGNWFDLA SE56 661-685 NO: 106 SEQ IDNSWDVFGNWFDLASWIKYIQYGVYI SE57 673-697 NO: 107 SEQ IDASWIKYIQYGVYIVVGVILLRIVIY SE58 685-709 NO: 108 SEQ IDIVVGVILLRIVIYIVQMLAKLRQGY SE59 697-721 NO: 109 SEQ IDYIVQMLAKLRQGYRPVFSSPPSYFQ SE60 709-733 NO: 110 SEQ IDYRPVFSSPPSYFQQTHIQQDPALPT SE61 721-745 NO: 111 SEQ IDQQTHIQQDPALPTREGKERDGGEGG SE62 733-757 NO: 112 SEQ IDTREGKERDGGEGGGNSSWPWQIEYI SE63 745-769 NO: 113 SEQ IDGGNSSWPWQIEYIHFLIRQLIRLLT SE64 757-781 NO: 114 SEQ IDIHFLIRQLIRLLTWLFSNCRTLLSR SE65 769-793 NO: 115 SEQ IDTWLFSNCRTLLSRVYQILQPILQRL SE66 781-805 NO: 116 SEQ IDRVYQILQPILQRLSATLQRIREVLR SE67 793-817 NO: 117 SEQ IDLSATLQRIREVLRTELTYLQYGWSY SE68 805-829 NO: 118 SEQ IDRTELTYLQYGWSYFHEAVQAVWRSA SE69 817-841 NO: 119 SEQ IDYFHEAVQAVWRSATETLAGAWGDLW SE70 829-853 NO: 120 SEQ IDATETLAGAWGDLWETLRRGGRWILA SE71 841-865 NO: 121 SEQ IDWETLRRGGRWILAIPRRIRQGLELTLL SE72 853-877 NO: 122The cultures were incubated overnight at 37° C. in a 7% CO₂ humidifiedatmosphere. Cells from each well were gently removed, transferred to 5.0ml FACS test tubes and washed. One set of cells was stained withanti-CD3+ anti-CD4+. The other duplicate set was stained with anti-CD3+anti-CD8+ (see below). These cell surface stained cells were thenpermeabilized and stained for intracellular content of IFN-gamma usingan anti-IFN-gamma staining antibody using standard intracellularstaining protocols. Each stained cell population (about 10,000 cellsfrom each tube) was then analyzed using a FACS flow cytometer and thefrequency of CD3+ CD4+ and CD3+ CD8+ T cells synthesizing IFN-gamma wasdetermined. The negative and positive controls were utilized forbackground control and for positive control references. About 1000analyses were performed in this manner during this experiment.

The frequency of CD4+ T cells (y axis) that expressed IFN-gamma byspleen cells from the six groups of mice in response to pools of SIV envpeptide (9 pools) and SIV gag peptides (7 pools) were determined. Alsodetermined was the frequency of CD8+ T cells (y axis) that expressIFN-gamma by spleen cells from the same six groups of mice in responseto pools of SIV env peptide (9 pools) and SIV gag peptides (7 pools).Data were the mean value from 4 mice/group. Results of these initialstudies indicated that delipidated SIVmac251 at a dose of 10 ug or 1.0ug led to marked augmentation of the SIV specific humoral responses inpreviously primed BALB/c mice. Even a dose of 0.1 ug (5×10⁶ viralparticles) led to detectable enhancement of the SIV specific humoralresponses in these mice. A dose of 1.0 ug, but not 10 ug, led tomarkedly broad breadth of SIV env and SIV gag peptide specific CD4+ Tcell responses as measured by IFN-g synthesis in previously primedBALB/c mice.

EXAMPLE 4

Charcoal Removal of Solvents after Plasma Delipidation

A charcoal column was generated by loading 2 ml of PBS-washed Hemasorbacharcoal into 3-ml BD LuerLock syringe containing a Whatman filter frit.The column was washed with 5% glucose/PBS (5 to 10 column volumes). Thecolumn was incubated in 5% glucose/PBS for 30 min. This column was usedto remove solvents from treated plasma.

About 2 ml of freshly isolated human plasma (ACD) was mixed with 1 ml ofone the following solvents: 1% DIPE; 10% DIPE; or butanol/DIPE (25:75).The mixture was vortexed for 15 seconds and then centrifuged 5 min at3000 rpm (˜1000×g). The solvent layer was aspirated. The plasma waspassed through the charcoal column described in the preceding paragraph.About 0.5 ml of PBS was used to wash the column. Washing may occurseveral times as needed. The results are shown in Table 9. Totalcholesterol (TC), triglycerides (TG), phospholipid (PL), apolipoproteinAl (ApoAl), apolipoprotein B (ApoB) and HDL were measured. The resultsshow good recoveries of ApoAl, ApoB and HDL compared to controls

TABLE 9 Analysis of Plasma Delipidated and Passed Through CharcoalSyringe Columns Sample TC TG PL ApoA1 ApoB HDL Assay CSI 132.8 83.5154.2 106.0 60.5 17.3 Assay CSII 197.4 164.7 224.0 87.2 107.7 21.3Control 1 66.2 76.0 57.4 47.8 31.5 10.5 Control 2 103.6 113.2 98.7 79.648.8 15.6 1% DIPE (1 pass) 48.1 59.2 38.6 39.4 15.6 10.1 1% DIPE (2pass) 40.1 46.5 29.3 26.0 15.7 6.8 10% DIPE (1 pass) 56.9 60.3 45.0 37.020.1 8.9 10% DIPE (2 pass) 58.9 61.7 52.4 42.2 25.5 8.5 But/DIPE (1pass) 57.4 65.0 54.1 47.7 24.8 9.1 But/DIPE (2 pass) 81.7 84.4 73.8 53.834.2 9.2Plasma Virus Recovery After Passage through Charcoal Column

Freshly isolated human plasma (ACD) was combined with HIV-1 to 1 ug/mlp24. HIV was added to the plasma such that the final concentration orparticle content was 1 ug/ml of virus p24 antigen. Next, 1 ml of thisplasma was passed through the column followed by 1 ml of PBS wash. Theflow through and wash were combined. This procedure was repeated twiceon fresh columns using 1 ml of the plasma. The flow through and washfrom each of these three runs were analyzed separately. The resultsshowed excellent recovery of p24 from the columns. P24 was measured by astandard capture ELISA protocol with a monoclonal antibody coated plate(for capture) and a polyclonal antibody for detection. Standard curveswith known amounts of p24 are used to determined the p24 content ofunknowns.

Direct Delipidation of HIV-1 and Removal of Solvents with CharcoalColumn and Retention of HIV Proteins

About 25 ul of 1000× HIV-1 IIIB was mixed with 1) nothing; 2) 12.5 ulbutanol/DIPE (25:75); 3) 2.5 ul 100% DIPE; or 4) 12.5 ul 1% DIPE in PBSand the samples were vortexed for 15 seconds. Charcoal columns (0.5-ml)were prepared as described above. The virus-solvent mixtures were loadedindividually onto separate columns. The columns were eluted with 1 ml ofPBS. The elution volumes were measured and samples assayed for p24 byELISA, protein, and subjected to Western blotting.

The samples treated with 1% DIPE showed excellent p24 recovery comparedto controls. The samples treated with 10% DIPE or butanol/DIPE showedslightly less p24 recovery. The total protein recovery was similar interms of percentage relative to control, to the p24 results obtained 1%DIPE, 10% DIPE or butanol/DIPE.

Western blot analysis, performed in a similar manner to the protocolprovided below in this example, revealed numerous immunoreactive bandswhen probed with human anti-HIV IgG with butanol/DIPE, 10% DIPE or 1%DIPE solvent treatments. Western blot analysis also revealed positiveimmunoreactive bands corresponding to p24 with butanol/DIPE, 10% DIPE or1% DIPE. Positive immunoreactive bands were observed for gp41 using 10%DIPE or 1% DIPE. Additional positive immunoreactive bands were observedfor gp120 with butanol/DIPE, 10% DIPE or 1%DIPE, although the intensityof staining was higher with 10% DIPE or 1% DIPE.

SIV and HIV Western Blot Analysis

Reagents for comparison included delipidated SIVmac251, heat inactivatedSIVmac251 and a rabbit polyclonal antibody against whole SIV (availablethrough the AIDS reagent repository, Rockville, Md.). About 1 ug ofprotein was required to visualize most of the SIV bands in the Westernblot. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed onthe viral lysates (lysate buffer:50 mM Tris-HCI, pH 7.4; 1% NP-40; 0.25%sodium deoxycholate; 150 mM NaCl; 1 mM EGTA; 1 mM PMSF; 1 ug/ml each ofaprotinin, leupeptin and pepstatin; 1 mM sodium vanadate; 1 mM NaF).

A silver stain was used to visualize the bands which reveal the variousviral proteins present following delipidation with respect to molecularweight standards. The heat inactivated SIVmac251 proteins were comparedwith the delipidated SIVmac251 proteins on the gels. A similar SDS-PAGEwas run and the proteins are transferred to nitrocellulose. The blottednitrocellulose was washed twice with water. A minimum of three blotseach for the delipidated SIVmac251 and the heat inactivated SIVmac251were run.

The blotted nitrocellulose was blocked in freshly prepared PBScontaining 3% nonfat dry milk (MLK) for 20 min at 20-25° C. withconstant agitation. The nitrocellulose strips were incubated with afreshly prepared pre-determined optimum concentration of the rabbitpolyclonal anti-SIV antiserum (about 5 ml of a 1:1000 dilution of theantiserum in PBS-MLK) overnight with agitation. The nitrocellulosestrips were washed twice with water. The strips were incubated withhorseradish peroxidases (HRP)-conjugated goat anti-rabbit IgG 1:3000dilution in PBS-MLK for 90 min at room temperature with agitation. Thenitrocellulose was washed with water twice and then with PBS-0.05% Tween20 for 3-5 min. The nitrocellulose strips were washed with 4-5 changesof water. Detection of the developed bands was achieved via detection ofthe developed bands. The bands developed using the heat inactivated SIVwith the delipidated SIV were compared.

A similar approach was used for Western blot analysis of solvent treatedHIV-1 passed through charcoal columns and probed for p24, gp41, gp120,and also for HIV antigens using an human anti-HIV IgG. Western blottingwas performed on SDS-PAGE separated virus samples transferred ontonitrocellulose membranes. The membranes are probed with polyclonal andmonoclonal antibodies to viral proteins and developed with secondaryantibodies conjugated with peroxidase and enhanced chemiluminescencereagents.

EXAMPLE 5 Use of a Kit for Delipidation of a Plasma Sample ContainingHIV and Production of Delipidated HIV Viral Particles

A 200 ml plasma sample, stored in a plasma bag with a tube connected toan opening in the bag, is obtained from blood drawn from a 22 year oldpatient afflicted with the human immunodeficiency virus (HIV) andshowing symptoms of acquired immunodeficiency syndrome (AIDS). Thepatient requires a reduction in the viral load in the blood. The plasmasample is exposed to a first extraction solvent to remove lipid from theviral envelope of the HIV virus.

A first container with a 500 ml capacity is removed from the kit. Thefirst container, which is graduated, contains a known volume (about 200ml) of a first extraction solvent. The entire plasma sample is added tothe first container through a removable screw cap. The first containeris agitated through repeated inversion, thereby mixing the firstextraction solvent and the plasma sample sufficiently to create amixture. The first container is placed on a counter and the mixturesettles into two phases.

The delipidated plasma phase is removed with a manual pipette or apipette connected to a vacuum, and placed in a second container from thekit. The volatile components of the first extraction solvent evaporate.Mild heating may be employed at this step. A tube, obtained from thekit, is inserted into the second container. The tube serves as, or isconnected to, an intravascular line leading to a needle introduced intothe antecubital vein of the patient. The delipidated fluid containingdelipidated plasma and delipidated HIV viral particles with reducedinfectivity is introduced into the vascular system through the force ofgravity by elevating the second container above the patient. The needleis optionally obtained from the kit. Administration of the delipidatedHIV viral particles into the vascular system induces an immune responsein the patient to epitopes on the delipidated HIV viral particles.

EXAMPLE 6 Use of a Kit for Delipidation of a Plasma Sample ContainingHIV and Production of Delipidated HIV Viral Particles

A 200 ml plasma sample, stored in a plasma bag with a tube connected toan opening in the bag, is obtained from blood drawn from a 22 year oldpatient afflicted with the human immunodeficiency virus (HIV) andshowing symptoms of AIDS. The patient requires a reduction in the viralload in the blood. The plasma sample is exposed to a first extractionsolvent to remove lipid from the viral envelope of the HIV virus.

A first container with a 500 ml capacity is removed from the kit. Thefirst container, which is graduated, contains a known volume (about 200ml) of a first extraction solvent. The entire plasma sample is added toa second container through a removable screw cap. The contents of thefirst container and the second container are added to a third containerobtained from the kit. The third container is agitated through repeatedinversion, thereby mixing the first extraction solvent and the plasmasample sufficiently to create a mixture. The third container is placedon a counter and the mixture settles into two phases.

The delipidated plasma phase is removed with a manual pipette or apipette connected to a vacuum, and placed in a fourth container from thekit. The volatile components of the first extraction solvent evaporate.Mild heating may be employed at this step. A tube, obtained from thekit, is inserted into the fourth container. The tube serves as, or isconnected to, an intravascular line leading to a needle introduced intothe antecubital vein of the patient. The delipidated fluid containingdelipidated plasma and delipidated HIV viral particles with reducedinfectivity is introduced into the vascular system through the force ofgravity by elevating the fourth container above the patient. The needleis optionally obtained from the kit. Administration of the delipidatedHIV viral particles into the vascular system induces an immune responsein the patient to epitopes on the delipidated HIV viral particles.

EXAMPLE 7 Use of a Kit for Delipidation of a Plasma Sample ContainingHIV and Production of Delipidated HIV Viral Particles

A 200 ml plasma sample, stored in a plasma bag with a tube connected toan opening in the bag, is obtained from blood drawn from a 22 year oldpatient afflicted with the human immunodeficiency virus (HIV) andshowing symptoms of AIDS. The patient requires a reduction in the viralload in the blood. The plasma sample is exposed to a first extractionsolvent to remove lipid from the viral envelope of the HIV virus.

A first container with a 500 ml capacity is removed from the kit. Thefirst container, which is graduated, contains a known volume (about 200ml) of a first extraction solvent. The entire plasma sample is added toa second container through a removable screw cap. The contents of thefirst container and the second container are added to a third containerobtained from the kit. The third container is agitated through repeatedinversion, thereby mixing the first extraction solvent and the plasmasample sufficiently to create a mixture. The third container is placedon a counter and the mixture settles into two phases.

The delipidated plasma phase is removed with a manual pipette or apipette connected to a vacuum, and placed in a fourth container from thekit. The volatile components of the first extraction solvent evaporate.Mild heating may be employed at this step. A second extraction solvent,contained in a graduated fifth container, is poured into the fourthcontainer in order to remove residual first extraction solvent. Thefourth container is agitated through repeated inversion, thereby mixingresidual first extraction solvent, the partially delipidated plasmasample and the second extraction solvent sufficiently to create amixture. The fourth container is allowed to sit and the mixtureseparates into a delipidated plasma layer and a solvent layer containingthe second extraction solvent and residual first extraction solvent. Thedelipidated plasma layer is removed and placed in a sixth containerobtained from the kit. A tube, obtained from the kit, is inserted intothe sixth container. The tube serves as or is connected to anintravascular line leading to a needle introduced into the antecubitalvein of the patient. The delipidated fluid containing delipidated plasmaand delipidated HIV viral particles with reduced infectivity isintroduced into the vascular system through the force of gravity byelevating the sixth container above the patient. The needle isoptionally obtained from the kit. Administration of the delipidated HIVviral particles into the vascular system induces an immune response inthe patient to epitopes on the delipidated HIV viral particles.

All patents, publications and abstracts cited above are incorporatedherein by reference in their entirety. It should be understood, ofcourse, that the foregoing relates only to preferred embodiments of thepresent invention and that numerous modifications or alterations may bemade therein without departing from the spirit and the scope of theinvention as set forth in the appended claims.

1-10. (canceled)
 11. A method for provoking a positive immune responsein an animal or human having a plurality of lipid-containing viralparticles comprising the steps of: obtaining a fluid containing thelipid-containing viral particles from the animal or the human;contacting the fluid containing the lipid-containing viral particleswith a first organic solvent capable of extracting lipid from thelipid-containing viral particles; mixing the fluid and the first organicsolvent; permitting organic and aqueous phases to separate; collectingthe aqueous phase containing modified viral particles with reduced lipidcontent; and introducing the aqueous phase containing the modified viralparticles with reduced lipid content into the animal or the humanwherein the modified viral particles with reduced lipid content provokea positive immune response in the animal or the human.
 12. The method ofclaim 11, further comprising: contacting the aqueous phase with ade-emulsifying agent capable of removing the first organic solvent; and,separating the de-emulsifying agent containing the removed first organicsolvent from the contacted aqueous phase.
 13. The method of claim 11,wherein after the aqueous phase is collected, the aqueous phase iscontacted with a de-emulsifying agent capable of removing the firstorganic solvent, and the de-emulsifying agent containing the removedfirst organic solvent is removed from the aqueous phase beforeintroducing the aqueous phase containing the modified viral particleswith reduced lipid content into the animal or the human.
 14. A methodfor treating a viral infection in an animal or human patient comprising:removing blood containing a plurality of lipid-containing infectiousviral particles from the animal or the human; obtaining plasma from theblood, the plasma containing the lipid-containing infectious viralparticles; contacting the plasma containing the lipid-containinginfectious viral particles with a first organic solvent capable ofextracting lipid from the lipid-containing infectious viral particles toproduce modified viral particles having reduced lipid content; mixingthe plasma and the first organic solvent; permitting organic and aqueousphases to separate; collecting the aqueous phase containing the modifiedviral particles; and introducing the aqueous phase containing themodified viral particles into the animal or the human wherein themodified viral particles have at least one exposed patient-specificantigen that was not exposed in the plurality of lipid-containinginfectious viral particles.
 15. The method of claim 14, wherein afterthe aqueous phase is collected, the aqueous phase is contacted with ade-emulsifying agent capable of removing the first organic solvent, andthe de-emulsifying agent containing the removed first organic solventfrom the contacted aqueous phase is separated and removed beforeintroducing the aqueous phase containing the modified viral particlesinto the animal or the human.
 16. The method of claim 14, furthercomprising adding cells to the aqueous phase containing the modifiedviral particles before introduction into the animal or the human. 17.The method of claim 15, further comprising adding cells to the aqueousphase containing the modified viral particles before introduction intothe animal or the human. 18-19. (canceled)
 20. A method of providingprotection in an animal or a human against a viral infection comprisingthe step of administering to the animal or the human of an effectiveamount of a composition comprising modified viral particles with reducedlipid content and a pharmaceutically acceptable carrier, wherein theamount is effective to provide a protective effect against infection bythe lipid-containing viral particle in the animal or the human.
 21. Themethod of claim 20 further comprising administration of animmunostimulant.
 22. The method of claim 11, wherein the first organicsolvent is an alcohol, an ether, an amine, a hydrocarbon, or acombination thereof.
 23. The method of 11, wherein the first organicsolvent is an alcohol, an ether, or a combination thereof.
 24. Themethod of claim 23 wherein the ether is C4 to C8 ether and the alcoholis a C1 to C8 alcohol.
 25. The method of 11, wherein the de-emulsifyingagent is an ether.
 26. The method of claim 11, wherein the fluid isplasma, serum, peritoneal fluid, lymphatic fluid, pleural fluid,pericardial fluid, cerebrospinal fluid, or a fluid of the reproductivesystem.
 27. The method of claim 11, wherein the first organic solvent isan alcohol, an ether, an amine, a hydrocarbon, an ester, a surfactant ora combination thereof.